Method for treating prostate cancer and/or gastrointestinal cancer

ABSTRACT

Gold(III) complexes having mixed ligands as anticancer agents. The gold(III) complexes are coordinated to bidentate ligands having diamino functional groups: a diaminocyclohexane ligand and an ethylenediamine ligand. These complexes can exist in both cis- and trans-configurations. Also described are pharmaceutical compositions incorporating the gold(I) complexes, methods of synthesis, methods of treating cancer and methods of inhibiting cancer cell proliferation and inducing cancer cell apoptosis

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 14/798,071, now allowed.

STATEMENT OF FUNDING ACKNOWLEDGEMENT

This project was funded by the National Plan for Science, Technology andInnovation (MAARIFAH)—King Abdulaziz City for Science and Technology—theKingdom of Saudi Arabia, award number (10-BIO1368-04).

BACKGROUND OF THE INVENTION

Technical Field

The present invention relates to pharmaceutical compounds. Moreparticularly, the present invention relates to gold(III) complexeshaving mixed diamine ligands. The present invention includes the use ofthese gold(III) complexes for treatment of cancers and cellproliferative disorders.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

The development of new metallodrugs with a pharmacological activitydifferent from platinum drugs is one of the major goals of modernbioinorganic and bio-organometallic medicinal chemistry research[Jankovic S M, Djekovic A, Bugarcic Z D, Jankovic S V, Lukic G, Folic M,Canovic D (2012) Effects of aurothiomalate and gold(III) complexes onspontaneous motility of isolated human oviduct. Biometals 25:919-925;Arsenijevic N, Volarevic V, Milovanovic M, Bugarcic Z D (2013) Gold(III)complexes, cytotoxic effects. In: Kretsinger R H, Uversky V N, PermyakovE A (eds) Encyclopedia of metalloproteins, vol 2. Springer, Heidelberg,pp 922-927; Kouroulis K N, Hadjikakou S K, Kourkoumelis N, Kubicki M,Male L, Hursthouse M, Skoulika S, Metsios A K, Tyurin V Y, Dolganov A B,Milaeva E R, Hadjiliadis N (2009) Synthesis, structural characterizationand in vitro cytotoxicity of new Au(III) and Au(I) complexes withthioamides. J Chem Soc Dalton Trans 47:10446-10456; Altaf M,Monim-ul-Mehboob M, Seliman A A, Isab A A, Dhuna V, Bhatia G, Dhuna K(2014) Synthesis, x-ray structures, spectroscopic analysis andanticancer activity of novel gold(I) carbene complexes. J Organomet Chem765:68-79; Hartinger C G, Dyson P J (2009) Bioorganometallicchemistry—from teaching paradigms to medicinal applications. Chem SocRev 38:391-401—each incorporated herein by reference in its entirety].Among these non-platinum anticancer drugs, gold complexes have recentlygained significant attention as a class of compounds with differentpharmacodynamic and kinetic properties than cisplatin with strong cellgrowth inhibiting effects [Kouroulis K N, Hadjikakou S K, KourkoumelisN, Kubicki M, Male L, Hursthouse M, Skoulika S, Metsios A K, Tyurin V Y,Dolganov A B, Milaeva E R, Hadjiliadis N (2009) Synthesis, structuralcharacterization and in vitro cytotoxicity of new Au(III) and Au(I)complexes with thioamides. J Chem Soc Dalton Trans 47:10446-10456; AltafM, Monim-ul-Mehboob M, Seliman A A, Isab A A, Dhuna V, Bhatia G, Dhuna K(2014) Synthesis, x-ray structures, spectroscopic analysis andanticancer activity of novel gold(I) carbene complexes. J Organomet Chem765:68-79—each incorporated herein by reference in its entirety]. Thecell growth inhibiting effects, in many cases, could be related toanti-mitochondrial effects that make the gold complexes interesting[Jankovic S M, Djekovic A, Bugarcic Z D, Jankovic S V, Lukic G, Folic M,Canovic D (2012) Effects of aurothiomalate and gold(I T) complexes onspontaneous motility of isolated human oviduct. Biometals 25:919-925;Arsenijevic N, Volarevic V, Milovanovic M, Bugarcic Z D (2013) Gold(III)complexes, cytotoxic effects. In: Kretsinger R H, Uversky V N, PermyakovE A (eds) Encyclopedia of metalloproteins, vol 2. Springer, Heidelberg,pp 922-927; Kouroulis K N, Hadjikakou S K, Kourkoumelis N, Kubicki M,Male L, Hursthouse M, Skoulika S, Metsios A K, Tyurin V Y, Dolganov A B,Milaeva E R, Hadjiliadis N (2009) Synthesis, structural characterizationand in vitro cytotoxicity of new Au(III) and Au(I) complexes withthioamides. J Chem Soc Dalton Trans 47:10446-10456—each incorporatedherein by reference in its entirety].

Oxaliplatin, the so-called third generation of platinum (II) complex wassynthesized as the most promising drug molecule in order to overcome thecrossresistance experienced by cisplatin [Graham J, Mushin M,Kirkpatrick P (2004) Fresh from thepipeline oxaliplatin. Nat Rev DrugDiscov 3(1):11-12—incorporated herein by reference in its entirety]. Itbears a 1,2-diaminocyclohexane (1,2-DACH) ligand and oxalate as aleaving group. The bulky chiral ligand, 1R,2R-diaminocyclohexane(1R,2R-DACH), contributes to high cytotoxicity againstcisplatin-resistant cell lines. It is possibly due to the sterichindrance effect of the 1,2-DACH-platinum-DNA adducts [Misset J L,Bleiberg H, Sutherland W, Bekradda M, Cvitkovic E (2000) Oxaliplatinclinical activity: a review. Crit Rev Oncol Hematol 35:75-93; ZdraveskiZ Z, Mello J A, Farinelli C K, Essigmann J M, Marinus M G (2002) MutSpreferentially recognizes cisplatin—over oxaliplatin—modified DNA. JBiol Chem 277:1255-1260—each incorporated herein by reference in itsentirety]. In the same line, several substituted 1,2-DACH complexes havebeen evaluated for their cytotoxicity [Chaney S G (1995) The chemistryand biology of platinum complexes with the 1,2-diaminocyclohexanecarrier ligand (review). Int J Oncol 6:1291-1305; Hoeschele J D,Showalter H D, Kraker A J, Elliott W L, Roberts B J, Kampf J W (1994)Synthesis, structural characterization, and antitumor properties of anovel class of large-ring platinum(II) chelate complexes incorporatingthe cis-1,4-diaminocyclohexane ligand in a unique locked boatconformation. J Med Chem 37:2630-2636—each incorporated herein byreference in its entirety]. Furthermore, a great number of Pt(II)complexes containing 1R,2R-DACH moiety have been synthesized and testedfor anticancer activities against a panel of human cancer lines. A fewof them have entered preclinical and clinical trials [Yu C W, Li K K,Pang S K, Au-Yeung S C, Ho Y P (2006) Anticancer activity of a series ofplatinum complexes integrating demethylcantharidin with isomers of1,2-diaminocyclohexane. Bioorg Med Chem Lett 16:1686-1691; Yu Y, Lou L,Liu W, Zhu H, Ye Q, Chen X, Gao W, Hou S (2008) Synthesis and anticanceractivity of lipophilic platinum(II) complexes of3,5-diisopropylsalicylate. Eur J Med Chem 43:1438-1443—each incorporatedherein by reference in its entirety]. Moreover, in search for betterplatinum(II) compounds, a wide variety of carrying ligands and leavinggroups have been screened. Monti E, Gariboldi M, Maiocchi A, Marengo E,Cassino C, Gabano E, Osella D (2005) Cytotoxicity of platinum(ii)conjugate models. The effect of chelating arms and leaving groups oncytotoxicity: a QSAR approach. J Med Chem 48:857-866; Berger 1, NazarovA A, Hartinger C G, Groessl M, Valiahdi S M, Jakupec M A, Keppler B K(2007), A glucose derivative as natural alternative to thecyclohexane-1,2-diamine ligand in the anticancer drug oxaliplatin. ChemMed Chem 2:505-514—each incorporated herein by reference in itsentirety].

Gold(III) complexes, which are isoelectronic and isostructural toplatinum(II) complexes, hold promise as possible anticancer agents[Chaves J D S, Neumann F, Francisco T M, Corrêa C C, Lopes M T P, SilvaH, Fontes A P S, de Almeida M V (2014), Synthesis and cytotoxic activityof gold(I) complexes containing phosphines and3-benzyl-1,3-thiazolidine-2-thione or 5-phenyl-1,3,4-oxadiazole-2-thioneas ligands, Inorg Chim Acta 414:85-90; Cutillas N, Yellol G S, de HaroC, Vicente C, Rodriguez V, Ruiz J (2013), Anticancer cyclometalatedcomplexes of platinum group metals and gold, Coord Chem Rev257:2784-2797—each incorporated herein by reference in its entirety].Surprisingly, only a few reports exist in the literature unfolding thecytotoxic properties and the in vivo anticancer effects of gold(III)complexes [van Rijt S H, Sadler P J (2009), Current applications andfuture potential for bioinorganic chemistry in the development ofanticancer drugs, Drug Discov Today 14(23-24): 1089-1097; Ronconi L,Marzano C, Zanello P, Corsini M, Miolo G, Macca C, Trevisan A, Fregona D(2006), Gold(III) dithiocarbamate derivatives for the treatment ofcancer: solution chemistry, DNA binding, and hemolytic properties. J MedChem 49:1648-1657—each incorporated herein by reference in itsentirety]. Gold(III) complexes having the same square-planar geometriesas cisplatin [Zou T, Lum C T, Chui S S, Che C-M (2013) Gold(III)complexes containing N-heterocyclic carbene ligalnds: thiol “Switchon”fluorescent probes and anti-cancer agents. Angew Chem 125:3002-3005;Cattaruzza L, Fregona D, Mongiat M, Ronconi M, Fassina A, Colombatti A,Aldinucci D (2011) Antitumor activity of gold(III)-dithiocarbamatoderivatives on prostate cancer cells and xenografts. Int J Cancer128(1):206-215—each incorporated herein by reference in its entirety],gold(III) complexes currently became the subject of profound anti-cancerresearch and hold great potential to enter clinical trials since some ofthem are highly cytotoxic to solid cancer tumors in vitro and in vivowhile causing minimal systemic toxicity [Ronconi L, Aldinucci D, Dou Q PD (2010) Latest insights into the anticancer activity ofgold(III)-dithiocarbamato complexes. Anticancer Agents Med Chem10:283-292; Sun R W Y, Che C M (2009) The anti-cancer properties ofgold(II) compounds with dianionic porphyrin and tetradentate ligands.Coord Chem Rev 253:1682-1691—each incorporated herein by reference inits entirety]. In general, gold(III) complexes are not very stable underphysiological conditions due to their high reduction potential and fasthydrolysis rate. Therefore, the selection of a suitable ligand toenhance the stability is a challenge in the design of gold(III)complexes. Au(III) is most likely coordinated by at least two chelatingnitrogen donors which lower the reduction potential of gold(III) centerand by this means stabilize the complex [Giovagnini L, Ronconi L,Aldinucci D, Lorenzon D, Sitran S, Fregoni D J (2005) Synthesis,characterization, and comparative in vitro cytotoxicity studies ofplatinum(II), palladium(II), and gold(III)methylsarcosinedithiocarbamate complexes. J Med Chem 48:1588-1592;Casini A, Hartinger C, Gabbiani C, Mini E, Dyson P J, Keppler B K,Messori L (2008) Gold(III) compounds as anticancer agents: Relevance ofgold-protein interactions for their mechanism of action. J Inorg Biochem102:564-575—each incorporated herein by reference in its entirety] andfacilitated extensive pharmacological investigation, both in vitro andin vivo [Tiekink E R T (2008) Anti-cancer potential of gold complexes.Inflammopharmacology 16:138-142; Casini A, Kelter G, Gabbiani C, CinelluM A, Minghetti G, Fregona D, Fiebig H H, Messori L (2009) Chemistry,antiproliferative properties, tumor selectivity, and molecularmechanisms of novel gold(III) compounds for cancer treatment: asystematic study. J Biol Inorg Chem 14:1139-1149—each incorporatedherein by reference in its entirety].

1,2-DACH ligand has structurally two asymmetric carbon centers, thus,1,2-DACH can exist as three isomeric forms which includes twoenantiomers (1R,2R-DACH) or (trans-1,2-DACH), (1S,2S-DACH) or(trans-1,2-DACH) and one diastereoisomer (1R,2S-DACH) or (cis-1,2-DACH).Since 1,2-DACH is chiral, the significance of stereochemical issues hasbeen addressed by a number of investigators which affect thecytotoxicity of complexes containing 1,2-DACH [Kidani Y, Inagaki K,Saito R, Tsukagoshi S (1977) Synthesis and anti-tumor activities ofplatinum(T) complexes of 1,2-diaminocyclohexane isomers and theirrelated derivatives. J Clin Hematol Oncol 7:197-202; Kemp S, Wheate N J,Buck D P, Nikac M, Collins J G, Aldrich-Wright J R (2007), The effect ofancillary ligand chirality and phenanthroline functional groupsubstitution on the cytotoxicity of platinum(II)-basedmetallointercalators. J Inorg Biochem 101:1049-1058—each incorporatedherein by reference in its entirety]. In spite of conflicting views[Gulloti M, Pasini A, Ugo R, Filippeschi S, Marmonti L, Spreafico F(1984) NMR coalescence effects resulting from stereochemicalnon-rigidity and halide exchange in octahedral rhodium(III) andiridium(III) tertiary phosphine complexes. Inorg Chim Acta 91:223-227;Noji M, Okamoto K, Kidani Y, Tashiro T (1981) Relation of conformationto antitumor activity of platinum(II) complexes of1,2-cyclohexanediamine and 2-(aminomethyl)cyclohexylamine isomersagainst leukemia P388. J Med Chem 24:508-515; Pasini A, Velcich A,Mariani A (1982) Absence of diastereoisomeric behaviour in theinteraction of chiral platinum anticancer compounds with DNA. Chem BiolInteract 42:311-320—each incorporated herein by reference in itsentirety], the consensus is that the (RR) isomer is generally moreactive than the (S,S) isomer [Burchenal J H, Kalaher K, O'Toole T,Chisholm J (1977), Lack of cross-resistance between certain platinumcoordination compounds in mouse leukemia. Cancer Res 37:3455-3457; BruckM A, Bau R, Noji M, Inagaki K, Kidani Y (1984), The crystal structuresand absolute configurations of the antitumor complexesPt(oxalato)(1R,2R-cyclohexanediamine) andPt(malonato)(1R,2R-cyclohexanediamine). Inorg Chim Acta 92:279-284—eachincorporated herein by reference in its entirety], although activity hasalso been demonstrated with the (R,S) isomer [Vollano J F, Al-Baker S,Dabrowiak J C, Schurig J E (1987) Comparative antitumor studies onplatinum(II) and platinum(IV) complexes containing1,2-diaminocyclohexane. J Med Chem 30:716-719—incorporated herein byreference in its entirety]. With regard to the stereochemistry of thecomplexes, Pt(II)(1R,2R-DACH) and Pt(II)(1S,2S-DACH) complexes have ahigher anticancer activity than Pt(1R,2S-DACH) complex [Johnson N P,Butour J L, Villani G, Wimmer F L, Defais M, Pierson V, Brabec V (1989)Metal antitumor compounds: the mechanism of action of platinumcomplexes. Prog Clin Biochem Med 10:1-24—incorporated herein byreference in its entirety]. However, the analogous gold(III) compound,[Au(en)₂]Cl₃ has been reported to have higher anticancer activity thangold(III) (1R,2R-DACH) (trans-1,2-DACH) and gold(III) (1S,2S-DACH)(trans-DACH) [Isab A A, Shaikh M N, Monim-ul-Mehboob M, Al-Maythalony BA, Wazeer M I M, Altuwaijri S (2011) Synthesis, characterization andanti proliferative effect of [Au(en)₂]Cl₃ and [Au(N-propyl-en)₂]Cl₃ onhuman cancer cell lines. Spectrochim Acta (A) 79:1196-1201;Monim-ul-Mehboob M, Altaf M, Fettouhi M, Isab A A, Wazeer M I M, ShaikhM N, Altuwaijri S (2013) Synthesis, spectroscopic characterization andanti-cancer properties of new gold(III)-alkanediamine complexes againstgastric, prostate and ovarian cancer cells; crystal structure of[Au₂(pn)₂(Cl)₂]Cl₂.H₂O. Polyhedron 61:225-23; Al-Maythalony B A, WazeerM I M, Isab A A (2009) Synthesis and characterization of gold(III)complexes with alkyldiamine ligands. Inorg Chim Acta 362:3109-3113;Al-Jaroudi S S, Fettouhi M, Wazeer M I M, Isab A A, Altuwaijri S (2013)Synthesis, characterization and cytotoxicity of new gold(III) complexeswith 1,2-diaminocyclohexane: influence of stereochemistry on antitumoractivity. Polyhedron 50:434-442; Al-Jaroudi S S, Monim-ul-Mehboob M,Altaf M, Fettouhi M, Wazeer M I M, Isab A A (2014) Synthesis,spectroscopic characterization, X-ray structure and electrochemistry ofnew bis(1,2-diaminocyclohexane) gold(III) chloride compounds and theiranticancer activities against PC3 and SGC7901 cancer cell lines. New JChem 38:3199-3211—each incorporated herein by reference in itsentirety].

As in the case of the parent cisplatin, the anticancer activity ofplatinum(II)-1,2-DACH is accompanied by toxicity. The emergence ofresistance, and low water solubility that can affect pharmacokinetics,are additional features that must be improved in the pursuit for a moreeffective material [Hanessian S, Wang J (1993) Hydrophilic analogs of(R,R)-diaminocyclohexane dichloroplatinum (DACH) and the influence ofrelative stereochemistry on antitumor activity. Can J Chem71:2102-2108—incorporated herein by reference in its entirety]. In viewof the foregoing, the present disclosure aims to provide gold(III)complexes having efficacy against a variety of cancers that also lackthe severe toxic side effects associated with platinum-based drugs.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the present disclosure relates to agold(III) complex having Formula A:

or a pharmaceutically acceptable salt, ester, solvate or prodrugthereof. The complex has a cis- or trans-configuration. 1-6 eachrepresents a carbon atom. R₁-R₈ are each independently selected from thegroup consisting of a hydrogen atom; a linear or branched, substitutedor unsubstituted C₁-C₈ alkyl group; and a substituted or unsubstitutedC₆-C₈ aryl group. R₉-R₂₀ are each independently selected from the groupconsisting of a hydrogen atom, a halogen atom, a hydroxyl group, aN-monosubstituted amino group, a N,N-disubstituted amino group, asubstituted or unsubstituted C₁-C₈ alkyl group, a substituted orunsubstituted C₁-C₈ alkoxy group, a substituted or unsubstituted C₃-C₈cycloalkyl group, and a substituted or unsubstituted C₆-C₈ aryl group.

In some embodiments, R₁-R₈ are each independently selected from thegroup consisting of a hydrogen atom, a substituted or unsubstitutedmethyl group, a substituted or unsubstituted ethyl group, a substitutedor unsubstituted propyl group, a substituted or unsubstituted isopropylgroup, a substituted or unsubstituted n-butyl group, a substituted orunsubstituted isobutyl group, a substituted or unsubstituted sec-butylgroup, a substituted or unsubstituted tert-butyl group, a substituted orunsubstituted n-pentyl group, a substituted or unsubstituted neopentylgroup, a substituted or unsubstituted sec-pentyl group, a substituted orunsubstituted tert-pentyl group, a substituted or unsubstituted n-hexanegroup, a substituted or unsubstituted isohexane group, and a substitutedor unsubstituted neohexane group. R₉-R₂₀ are each independently ahydrogen atom, a halogen atom, a N-monosubstituted amino group, aN,N-disubstituted amino group, a substituted or unsubstituted methylgroup, a substituted or unsubstituted ethyl group, a substituted orunsubstituted propyl group, a substituted or unsubstituted isopropylgroup, a substituted or unsubstituted n-butyl group, a substituted orunsubstituted isobutyl group, a substituted or unsubstituted sec-butylgroup, a substituted or unsubstituted tert-butyl group, a substituted orunsubstituted n-pentyl group, a substituted or unsubstituted neopentylgroup, a substituted or unsubstituted sec-pentyl group, a substituted orunsubstituted tert-pentyl group, a substituted or unsubstituted n-hexanegroup, a substituted or unsubstituted isohexane group, and a substitutedor unsubstituted neohexane group.

In certain embodiments, the gold(III) complex has a formula selectedfrom the group consisting of Formula 1a, Formula 1b, Formula 2a andFormula 2b:

In one or more embodiments, the gold(III) complex further comprises oneor more pharmaceutically acceptable anions selected from the groupconsisting of fluoride, chloride, bromide, iodide, nitrate, sulfate,phosphate, amide, methanesulfonate, ethanesulfonate, p-toluenesulfonate,salicylate, malate, maleate, succinate, tartarate, citrate, acetate,perchlorate, trifluoromethanesulfonate, acetylacetonate,hexafluorophosphate, and hexafluoroacetylacetonate.

According to a second aspect, the present disclosure relates to acomposition comprising the gold(III) complex of according to the firstaspect or a pharmaceutically acceptable salt, ester, solvate or prodrugthereof; and one or more pharmaceutically acceptable carriers.

In one embodiment, the composition further comprises one or more otheractive pharmaceutical agents.

In one embodiment, the composition is in solid, semi-solid or liquiddosage forms.

In one or embodiments, the composition is formulated for one or moremodes of administration selected from the group consisting of oraladministration, systemic administration, parenteral administration,inhalation spray, infusion rectal administration, topicaladministration, intravesical administration, intradermal administration,transdermal administration, subcutaneous administration, intramuscularadministration, intralesional administration, intracranialadministration, intrapulmonal administration, intracardialadministration, intrasternal administration and sublingualadministration.

In a third aspect, the present disclosure is directed to a method fortreating one or more types of cancer in a mammalian subject in needthereof. The method comprises administering a therapeutically effectiveamount of the composition of the second aspect to the mammalian subject.

In one embodiment, the one or more types of cancer are prostate cancerand/or gastrointestinal cancer.

In one embodiment, the therapeutically effective amount comprises 5-50μM of the gold(III) complex or a pharmaceutically acceptable salt,ester, solvate or prodrug thereof.

In a fourth aspect, the present disclosure provides a method forinhibiting proliferation of cancer cells. The method comprise contactingthe cancer cells with the gold(III) complex of the first aspect or apharmaceutically acceptable salt, ester, solvate or prodrug thereof.

In at least one embodiment, the cancer cells are human cells.

In some embodiments, the cancer cells are prostate cancer cells and/orgastrointestinal cancer cells.

In some embodiments, the gold(III) complex has a concentration of 5-50μM.

In one embodiment, the gold(III) complex exhibits an IC₅₀ of 1-20 μM forinhibiting the proliferation of the prostate cancer cells and/or thegastrointestinal cancer cells.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIGS. 1A and 1B are bar graphs showing the time-dependentantiproliferative effect of 10 μM Compound 1 on PC3 and SGC7901 cells,respectively, for 24 and 72 h using MTT assay where results wereexpressed as the mean, SD, *P<0.05.

FIGS. 2A and 2B are bar graphs showing the time-dependentantiproliferative effect of 10 μM Compound 2 on PC3 and SGC7901 cells,respectively, for 24 and 72 h using MTT assay where results wereexpressed as the mean, SD, *P<0.05.

FIGS. 3A and 3B are bar graphs showing the time-dependentantiproliferative effect of 310 μM Compound 3 on PC3 and SGC7901 cells,respectively, for 24 and 72 h using MTT assay where results wereexpressed as the mean, SD, *P<0.05.

FIGS. 4A and 4B are bar graphs showing the concentration-dependentantiproliferative effect of 10 μM Compound 1 on PC3 and SGC7901 cells,respectively, for 24 and 72 h using MTT assay where results wereexpressed as the mean, SD, *P<0.05.

FIGS. 5A and 5B are bar graphs showing the concentration-dependentantiproliferative effect of 10 μM Compound 2 on PC3 and SGC7901 cells,respectively, for 24 and 72 h using MTT assay where results wereexpressed as the mean, SD, *P<0.05.

FIGS. 6A and 6B are bar graphs showing the concentration-dependentantiproliferative effect of 310 μM Compound 3 on PC3 and SGC7901 cells,respectively, for 24 and 72 h using MTT assay where results wereexpressed as the mean, SD, *P<0.05.

FIGS. 7A and 7B are bar graphs comparing the time-dependentantiproliferative effect of 10 μM Compounds 1, 2 and 3 on PC3 andSGC7901 cells, respectively, for 24 and 72 h using MTT assay whereresults were expressed as the mean, SD, *P<0.05.

FIGS. 8A, 8B and 8C show UV-Vis spectra of Compounds 1, 2 and 3,respectively, followed by dissolution in the buffer solution at 37° C.(a) just after mixing and (b) after 7 days.

FIG. 9 is a solution state ¹H NMR spectrum of[{(S,S)-(+)-(1,2-DACH)}Au(en)]Cl₃ complex.

FIG. 10 is a solution state ¹³C{¹H} NMR spectrum of[{(S,S)-(+)-(1,2-DACH)}Au(en)]Cl₃ complex.

FIGS. 11A, 11B, 11C and 11D are geometries of 1(a), 1(b), 2(a) and 2(b),respectively, obtained at the B3LYP/LanL2DZ level of theory usingGAUSSIAN09.

FIGS. 12A and 12B are solution state ¹H NMR spectra of[{(cis)-(+)-(1,2-DACH)}Au(en)]Cl₃ complex in D₂O at after 7 days andjust after mixing, respectively.

FIGS. 13A and 13B are solution state ¹³C{¹H} NMR spectra of[{(cis)-(+)-(1,2-DACH)}Au(en)]Cl₃ complex in D₂O at after 7 days andjust after mixing, respectively.

FIGS. 14A and 14B are solution state ¹H NMR spectra of[{(S,S)-(+)-(1,2-DACH)}Au(en)]Cl₃ complex in D₂O at after 7 days andjust after mixing, respectively.

FIGS. 15A and 15B are solution state ¹³C{¹H} NMR spectra of[{(S,S)-(+)-(1,2-DACH)}Au(en)]Cl₃ complex in D₂O at after 7 days andjust after mixing, respectively.

FIGS. 16A, 16B and 16C are cyclic voltammograms of Compounds 1, 2 and 3,respectively, in the phosphate buffer at platinum electrode.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views.

The present disclosure will be better understood with reference to thefollowing definitions:

As used herein, “compound” and “complex” are used interchangeably, andare intended to refer to a chemical entity, whether in the solid, liquidor gaseous phase, and whether in a crude mixture or purified andisolated.

The term “alkyl”, as used herein, unless otherwise specified, refers toa saturated straight, branched, or cyclic, primary, secondary, ortertiary hydrocarbon of typically C₁ to C₈, and specifically includesmethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, 1-butyl,pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The term optionally includes substituted alkylgroups. Moieties with which the alkyl group can be substituted areselected from the group consisting of hydroxyl, amino, alkylamino,arylamino, alkoxy, thioalkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., “Protective Groups in OrganicSynthesis”, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference in its entirety.

The term “aryl”, as used herein, and unless otherwise specified, refersto phenyl, biphenyl, or naphthyl, and preferably phenyl. The termincludes both substituted and unsubstituted moieties. The aryl group canbe substituted with one or more moieties selected from the groupconsisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy,nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, orphosphonate, either unprotected, or protected as necessary, as known tothose skilled in the art, for example, as taught in Greene, et al.,“Protective Groups in Organic Synthesis”, John Wiley and Sons, SecondEdition, 1991, hereby incorporated by reference in its entirety.

As used herein, “analogue” refers to a chemical compound that isstructurally similar to a parent compound, but differs slightly incomposition (e.g., one atom or functional group is different, added, orremoved). The analogue may or may not have different chemical orphysical properties than the original compound and may or may not haveimproved biological and/or chemical activity. For example, the analoguemay be more hydrophilic or it may have altered reactivity as compared tothe parent compound. The analogue may mimic the chemical and/orbiologically activity of the parent compound (i.e., it may have similaror identical activity), or, in some cases, may have increased ordecreased activity. The analogue may be a naturally or non-naturallyoccurring variant of the original compound. Other types of analoguesinclude isomers (enantiomers, diastereomers, and the like) and othertypes of chiral variants of a compound, as well as structural isomers.

As used herein, “derivative” refers to a chemically or biologicallymodified version of a chemical compound that is structurally similar toa parent compound and (actually or theoretically) derivable from thatparent compound. A “derivative” differs from an “analogue” in that aparent compound may be the starting material to generate a “derivative,”whereas the parent compound may not necessarily be used as the startingmaterial to generate an “analogue.” A derivative may or may not havedifferent chemical or physical properties of the parent compound. Forexample, the derivative may be more hydrophilic or it may have alteredreactivity as compared to the parent compound. Derivatization (i.e.,modification) may involve substitution of one or more moieties withinthe molecule (e.g., a change in functional group). The term “derivative”also includes conjugates, and prodrugs of a parent compound (i.e.,chemically modified derivatives which can be converted into the originalcompound under physiological conditions).

The term “prodrug” refers to an agent that is converted into abiologically active form in vivo. Prodrugs are often useful because, insome situations, they may be easier to administer than the parentcompound. They may, for instance, be bioavailable by oral administrationwhereas the parent compound is not. The prodrug may also have improvedsolubility in pharmaceutical compositions over the parent drug. Aprodrug may be converted into the parent drug by various mechanisms,including enzymatic processes and metabolic hydrolysis [Harper, N.J.(1962). Drug Latentiation in Jucker, ed. Progress in Drug Research,4:221-294; Morozowich et al (1977). Application of Physical OrganicPrinciples to Prodnig Design in E. B. Roche ed. Design ofBiopharmaceutical Properties through Prodrugs and Analogs, APhA; Acad.Pharm. Sci.; E. B. Roche, ed. (1977). Bioreversible Carriers in Drug inDrug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985)Design of Prodrugs, Elsevier; Wang et al. (1999) Prodrug approaches tothe improved delivery of peptide drug, Curr. Pharm. Design.5(4):265-287;Pauletti et al. (1997). Improvement in peptide bioavailability:Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev.27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs for OralDelivery of β-Lactam antibiotics, Pharm. Biotech. 11:345-365; Gaignaultet al. (1996). Designing Prodrugs and Bioprecursors I. Carrier Prodrugs,Pract. Med. Chem. 671-696; M. Asgharnejad (2000). Improving Oral DrugTransport Via Prodrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds.,Transport Processes in Pharmaceutical Systems, Marcell Dekker, p.185-218; Balant et al. (1990) Prodrugs for the improvement of drugabsorption via different routes of administration, Eur. J. Drug Metab.Pharmacokinet., 15(2): 143-53; Balimane and Sinko (1999). Involvement ofmultiple transporters in the oral absorption of nucleoside analogues,Adv. Drug Delivery Rev., 39(1-3): 183-209; Browne (1997). Fosphenyloin(Cerebyx), Clin. Neuropharmacol. 20(1): 1-12; Bundgaard (1979).Bioreversible derivatization of drugs—principle and applicability toimprove the therapeutic effects of drugs, Arch. Pharm. Chemi. 86(1):1-39, H. Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier;Fleisher et al. (1996). Improved oral drug delivery: solubilitylimitations overcome by the use of prodrugs, Adv. Drug Delivery Rev.19(2): 115-130; Fleisher et al. (1985). Design of prodrugs for improvedgastrointestinal absorption by intestinal enzyme targeting, MethodsEnzymol. 112: 360-81; Farquhar D, et al. (1983). Biologically ReversiblePhosphate-Protective Groups, J. Pharm. Sci., 72(3): 324-325; Han, H. K.et al. (2000). Targeted prodrug design to optimize drug delivery, AAPSPharm Sci., 2(1): E6, Sadzuka Y. (2000) Effective prodnrug liposome andconversion to active metabolite, Curr. Drug Melab., 1(1):31-48; D. M.Lambert (2000) Rationale and applications of lipids as prodrug carriers,Eur. J. Pharm. Sci., 11 Suppl 2:S15-27; Wang, W. et al. (1999) Prodrugapproaches to the improved delivery of peptide drugs. Curr. Pharm. Des.,5(4):265-87—each incorporated herein by reference in its entirety]. Insome embodiments, “Pharmaceutically acceptable prodrugs” refer to acompound that is metabolized, for example hydrolyzed or oxidized, in thehost to form the pharmaceutical composition of the present disclosure.Typical examples of prodrugs include compounds that have biologicallylabile protecting groups on a functional moiety of the active compound.Prodrugs include compounds that can be oxidized, reduced, aminated,deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed,alkylated, dealkylated, acylated, deacylated, phosphorylated,dephosphorylated to produce the active compound.

The term “solvate” means a physical association of a compound of thisdisclosure with one or more solvent molecules, whether organic orinorganic. This physical association includes hydrogen bonding. Incertain instances the solvate will be capable of isolation, for examplewhen one or more solvent molecules are incorporated in the crystallattice of the crystalline solid. The solvent molecules in the solvatemay be present in a regular arrangement and/or a non-orderedarrangement. The solvate may comprise either a stoichiometric ornonstoichiometric amount of the solvent molecules. Solvate encompassesboth solution-phase and isolable solvates. Exemplary solvates include,but are not limited to, hydrates, ethanolates, methanolates, andisopropanolates. Methods of solvation are generally known in the art.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to cancer or pathologies related to increased celldivision, a therapeutically effective amount refers to that amount whichhas the effect of at least one of the following: (1) reducing the sizeof a tumor, (2) inhibiting (that is, slowing to some extent, preferablystopping) aberrant cell division, growth or proliferation, for examplecancer cell division, (3) preventing or reducing the metastasis ofcancer cells, (4) relieving to some extent (or, preferably, eliminating)one or more symptoms associated with a pathology related to or caused inpart by unregulated or aberrant cellular division, including forexample, cancer and (5) inducing apoptosis of cancer cells or tumorcells.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in theprevention, treatment and/or management of a cancer or one or moresymptoms thereof.

As used herein, the terms “treat,” “treatment,” and “treating” in thecontext of the administration of a therapy to a subject in need thereofrefer to the reduction or inhibition of the progression and/or durationof cancer, the reduction or amelioration of the severity of cancer,and/or the amelioration of one or more symptoms thereof resulting fromthe administration of one or more therapies. In some embodiments, thesubject is a mammalian subject. In one embodiment, the subject is ahuman. “Treating” or “treatment” of a disease includes preventing thedisease from occurring in a subject that may be predisposed to thedisease but does not yet experience or exhibit symptoms of the disease(prophylactic treatment), inhibiting the disease (slowing or arrestingits development), providing relief from the symptoms or side-effects ofthe disease (including palliative treatment), and relieving the disease(causing regression of the disease). With regard to cancer orhyperplasia, these terms simply mean that the life expectancy of anindividual affected with a cancer will be increased or that one or moreof the symptoms of the disease will be reduced. In specific embodiments,such terms refer to one, two or three or more results following theadministration of one, two, three or more therapies: (1) astabilization, reduction or elimination of the cancer stem cellpopulation; (2) a stabilization, reduction or elimination in the cancercell population; (3) a stabilization or reduction in the growth of atumor or neoplasm; (4) an impairment in the formation of a tumor; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalization rate, (10) a decrease in hospitalization lengths, (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%, and (12) an increase in the number of patientsin remission. In certain embodiments, such terms refer to astabilization or reduction in the cancer stem cell population. In someembodiments, such terms refer to a stabilization or reduction in thegrowth of cancer cells. In some embodiments, such terms refer tostabilization or reduction in the cancer stem cell population and areduction in the cancer cell population. In some embodiments, such termsrefer to a stabilization or reduction in the growth and/or formation ofa tumor. In some embodiments, such terms refer to the eradication,removal, or control of primary, regional, or metastatic cancer (e.g.,the minimization or delay of the spread of cancer). In some embodiments,such terms refer to a reduction in mortality and/or an increase insurvival rate of a patient population. In further embodiments, suchterms refer to an increase in the response rate, the durability ofresponse, or number of patients who respond or are in remission. In someembodiments, such terms refer to a decrease in hospitalization rate of apatient population and/or a decrease in hospitalization length for apatient population.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Therefore, thepharmaceutical composition refers to the combination of an active agentwith a carrier, inert or active, making the composition especiallysuitable for diagnostic or therapeutic use in vivo or ex vivo.

“Pharmaceutically acceptable salt” or “pharmaceutically acceptableester” refers to a compound in a pharmaceutically acceptable form suchas an ester, a phosphate ester, a salt of an ester or a related) which,upon administration to a subject in need thereof, provides at least oneof the gold(I) complexes described herein. Pharmaceutically acceptablesalts and ester retain the biological effectiveness and properties ofthe free bases and which are obtained by reaction with inorganic ororganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,succinic acid, tartaric acid, citric acid, and the like. Suitable saltsinclude those derived from alkali metals such as potassium and sodium,alkaline earth metals such as calcium and magnesium, among numerousother acids well known in the art.

A “pharmaceutical composition” refers to a mixture of the compoundsdescribed herein or pharmaceutically acceptable salts, esters orprodrugs thereof, with other chemical components, such asphysiologically acceptable carriers and excipients. One purpose of apharmaceutical composition is to facilitate administration of at leastone gold(I) complex to a subject.

As used herein, a “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” refers to a carrier or diluentthat does not cause significant irritation to an organism and does notabrogate the biological activity and properties of the administeredgold(I) complex. The term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005,which is incorporated herein by reference in its entirety. Examples ofphysiologically acceptable carriers include buffers such as phosphatebuffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.). An “excipient” refersto an inert substance added to a pharmaceutical composition to furtherfacilitate administration of a compound. Examples, without limitation,of excipients include calcium carbonate, calcium phosphate, varioussugars and types of starch, cellulose derivatives, gelatin, vegetableoils and polyethylene glycols.

As used herein, a “binder” holds the ingredients in a tablet together.Binders ensure that tablets and granules can be formed with requiredmechanical strength, and give volume to low active dose tablets. Bindersmay be: (1) saccharides and their derivatives, such as sucrose, lactose,starches, cellulose or modified cellulose such as microcrystallinecellulose, carboxymethyl cellulose, and cellulose ethers such ashydroxypropyl cellulose (HPC), and sugar alcohols such as xylitol,sorbitol or maltitol (2) proteins such as gelatin and (3) syntheticpolymers including polyvinylpyrrolidone (PVP), polyethylene glycol(PEG). Binders are classified according to their application. Solutionbinders are dissolved in a solvent (for example water or alcohol can beused in wet granulation processes). Examples include gelatin, cellulose,cellulose derivatives, polyvinylpyrrolidone, starch, sucrose andpolyethylene glycol. Dry binders are added to the powder blend, eitherafter a wet granulation step, or as part of a direct powder compression(DC) formula. Examples include cellulose, methyl cellulose,polyvinylpyrrolidone and polyethylene glycol.

The terms “including”, “such as”, “for example” and the like areintended to refer to exemplary embodiments and not to limit the scope ofthe present disclosure.

Gold(III) Complexes and Pharmaceutical Compositions Thereof

The present disclosure provides gold(III) complexes having medicinal orpharmaceutical properties, preferably antitumor, anticancer and/orantiproliferative properties. In these gold(III) complexes, each centralgold(III) atom is coordinated, preferably chelated by two or more mixedor different ligands. Each ligand is diamine-based and contains twoamino groups as functional groups. Specifically, the ligands arediaminocyclohexane and ethylenediamine with their base, unsubstitutedstructures shown below:

Both the diaminocyclohexane and ethylenediamine ligands as well asderivatives thereof bind to the central gold(III) atom in a bidentatemanner. The nitrogen atoms in the diamino groups act as electron donoratoms that the gold(III) atom is coordinated. In other words, thegold(III) atom is coordinated to two donor nitrogen atoms from onediaminocyclohexane ligand and two donor nitrogen atoms from oneethylenediamine ligand. Accordingly, a gold(TIT) complex provided hereinhas a generic structure of, in cis-(1R,2S or 1S,2R) and trans-(1R,2R or1S,2S) configurations:

where:

-   -   1-6 each represents a carbon atom;    -   R₁-R₈ are each independently a hydrogen; a linear or branched,        substituted or unsubstituted C₁-C₈ alkyl group; or a substituted        or unsubstituted C₆-C₈ aryl group; and    -   R₉-R₂₀ are each independently selected from the group consisting        of a hydrogen atom, a halogen atom, a hydroxyl group a        N-monosubstituted amino group, a N,N-disubstituted amino group,        a substituted or unsubstituted C₁-C₈ alkyl group, a substituted        or unsubstituted C₁-C₈ alkoxy group a substituted or        unsubstituted C₃-C₈ cycloalkyl group, and a substituted or        unsubstituted C₆-C₈ aryl group.

In some embodiments, a gold(III) complex provided herein has a structureaccording to Formula A in cis-(1R,2S or 1S,2R) and trans-(1R,2R or1S,2S) configurations, where:

-   -   R₁-R₈ are each independently a hydrogen; or a substituted or        unsubstituted methyl, ethyl, propyl, isopropyl, n-butyl,        isobutyl, sec-butyl group, tert-butyl, n-pentyl, neopentyl,        sec-pentyl, tert-pentyl, n-hexane, isohexane, a neohexane group;        and    -   R₉-R₂₀ are each independently a hydrogen; a halogen; a        N-monosubstituted amino group; a N,N-disubstituted amino group;        or a substituted or unsubstituted methyl, ethyl, propyl,        isopropyl, n-butyl, isobutyl, sec-butyl group, tert-butyl,        n-pentyl, neopentyl, sec-pentyl, tert-pentyl, n-hexane,        isohexane, a neohexane group.

In one embodiment, the gold(III) complex of the present disclosure isone of the following:

-   -   cis-(1R,2S)-(1,2-diaminocyclohexane)gold(III) ethylenediamine;    -   cis-(1S,2R)-(1,2-diaminocyclohexane)gold(III) ethylenediamine;    -   trans-(1R,2R)-(−)-(1,2-diaminocyclohexane)gold(III)        ethylenediamine;    -   trans-(1S,2S)-(+)-(1,2-diaminocyclohexane)gold(III)        ethylenediamine;

In one embodiment, the gold(III) complex of the present disclosure isaccording to one of the Formulas 1a, 1b, 2a and 2b:

In certain embodiments, especially but not limited to pharmaceuticalapplications, the gold(III) complex can further include a counter-anionto form a pharmaceutically acceptable salt. As used herein, the term“counter-anion” refers to an anion, preferably a pharmaceuticallyacceptable anion that is associated with a positively charged gold(III)complex of at least one of the Formulas A, 1a, 1b, 2a and 2b.Non-limiting examples of pharmaceutically counter-anions include halidessuch as fluoride, chloride, bromide, iodide; nitrate; sulfate;phosphate; amide; methanesulfonate; ethanesulfonate; p-toluenesulfonate,salicylate, malate, maleate, succinate, tartarate; citrate; acetate;perchlorate; trifluoromethanesulfonate (triflate); acetylacetonate;hexafluorophosphate; and hexafluoroacetylacetonate. In some embodiments,the counter-anion is a halide, preferably chloride.

Another aspect of the present disclosure relates to pharmaceuticalcomposition comprising one or more of the mixed diamine ligand gold(III)complexes described herein. In other words, the gold(IIIl) complexesdescribed herein or analogues or derivatives thereof can be provided ina pharmaceutical composition. Depending on the intended mode ofadministration, the pharmaceutical composition can be in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include a therapeuticallyeffective amount of one or more of the gold(III) complexes describedherein or derivatives thereof in combination with a pharmaceuticallyacceptable carrier and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, diluents or other non-activeingredients. By pharmaceutically acceptable is meant a material that isnot biologically or otherwise undesirable, which can be administered toan individual along with the selected compound without causingsignificant unacceptable biological effects or interacting in adeleterious manner with the other components of the pharmaceuticalcomposition in which it is contained.

A mixed diamine ligand gold(III) complex of the present disclosure or ananalogue or derivative thereof may be used in conjunction with one ormore additional compounds, in the treatment or prevention of neoplasm;of tumor or cancer cell division, growth, proliferation and/ormetastasis in a mammalian subject; induction of death or apoptosis oftumor and/or cancer cells; and/or any other form of proliferativedisorder. A gold(III) complex of the present disclosure can beformulated as a pharmaceutical composition.

The neoplastic activity of the tumor or cancer cells may be localized orinitiated in one or more of the following: blood, brain, bladder, lung,cervix, ovary, colon, rectum, pancreas, skin, prostate gland, stomach,breast, liver, spleen, kidney, head, neck, testicle, bone (includingbone marrow), thyroid gland, central nervous system. The mixed diamineligand gold(III) complex of the present disclosure or the pharmaceuticalcomposition thereof is especially effective in the treatment orprevention of colorectal cancer (including colon cancer, rectum cancerand bowel cancer); lung cancer (including non-small cell lung carcinomaor NSCLC and small cell lung carcinoma); cervical cancer (including thehistologic subtypes of squamous cell carcinoma, adenocarcinoma,adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumor,glass cell carcinoma, villoglandular adenocarcinoma, melanoma andlymphoma).

A pharmaceutical composition comprising one or more gold(III) complexesof the present disclosure can then be administered orally, systemically,parenterally, by inhalation spray, rectally, or topically in dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. In someembodiments, the method of administration of the steroid or an analogueor derivative thereof is oral. In other embodiments, the compound or ananalogue or derivative thereof is administered by injection, such as,for example, through a peritumoral injection.

Topical administration can also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes intravesical, intradermal,transdermal, subcutaneous, intramuscular, intralesional, intracranial,intrapulmonal, intracardial, intrasternal and sublingual injections, orinfusion techniques. Formulation of drugs is discussed in, for example,Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa.; 1975. Another example of includes Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980, which is incorporated herein by reference in its entirety.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Dimethyl acetamide, surfactantsincluding ionic and non-ionic detergents, polyethylene glycols can beused. Mixtures of solvents and wetting agents such as those discussedabove are also useful. Suppositories for rectal administration of thecompound or an analogue or derivative thereof can be prepared by mixingthe steroid or an analogue or derivative thereof with a suitablenonirritating excipient such as cocoa butter, synthetic mono- di- ortriglycerides, fatty acids and polyethylene glycols that are solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum and release the drug.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms, thecompounds of this disclosure are ordinarily combined with one or moreadjuvants appropriate to the indicated route of administration. Ifadministered per os, a contemplated steroid or an analogue or derivativethereof can be admixed with lactose, sucrose, starch powder, celluloseesters of alkanoic acids, cellulose alkyl esters, talc, stearic acid,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets cancontain a controlled-release formulation as can be provided in adispersion of active compound in hydroxypropylmethyl cellulose. In thecase of capsules, tablets, and pills, the dosage forms can also comprisebuffering agents such as sodium citrate, magnesium or calcium carbonateor bicarbonate. Tablets and pills can additionally be prepared withenteric coatings.

For therapeutic purposes, formulations for parenteral administration canbe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. These solutions and suspensions can beprepared from sterile powders or granules having one or more of thecarriers or diluents mentioned for use in the formulations for oraladministration. A contemplated steroid or an analogue or derivativethereof of the present disclosure can be dissolved in water,polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseedoil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/orvarious buffers. Other adjuvants and modes of administration are welland widely known in the pharmaceutical art.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

The amount of active ingredients that can be combined with the carriermaterials to produce a single dosage form varies depending upon themammalian subject treated and the particular mode of administration.

Methods of Synthesis

The 1,2-(diaminocyclohexane)gold(III) ethylenediamine complexes of thepresent disclosure are not limited by their synthesis routes andmethods. These gold(III) complexes can be prepared by various previouslyreported synthesis protocols with slight modifications as recognized asappropriate by a person of ordinary skill in the pharmaceutical andmedicinal chemistry arts [U.S. Pat. No. 8,895,611; Al-Maythalony B A,Wazeer M I M, Isab A A (2009) Synthesis and characterization ofgold(III) complexes with alkyldiamine ligands. Inorg Chim Acta362:3109-3113; Al-Jaroudi S S, Fettouhi M, Wazeer M I M, Isab A A,Altuwaijri S (2013) Synthesis, characterization and cytotoxicity of newgold(III) complexes with 1,2-diaminocyclohexane: influence ofstereochemistry on antitumor activity. Polyhedron 50:434-442; Zhu S,Gorski W, Powell D R, Walmsley J A (2006) Synthesis, structures, andelectrochemistry of gold(III) ethylenediamine complexes and interactionswith guanosine 5′-monophosphate. Inorg Chem 45:2688-2694; Fernandez E J,Garcia-Luzuriaga E, Laguna A, Lopez-de-Luzuriaga, J M, Olmos, M E (2010)Synthesis of gold(III) complexes of 2-(diphenylthiophosphino)aniline;Casini A, Diawara, M C, Scopelliti R, Zakeeruddin S M, Gratzel M, DysonP J (2010) Synthesis, characterization and biological properties ofgold(TIT) compounds with modified bipyridine and bipyridylamine ligands.Dalton Trans. 39:2239-2245; Johnson M W, DiPasquale, A G, Bergman, R G,Toste, F D (2014) Synthesis of stable gold(III) pincer complexes withanionic heteroatom donors Organometallics 33:4169-4172; Moustatih A,Garnier-Suillerot A (1989) Bifunctional antitumor compounds: Synthesisand characterization of a gold(III)-streptonigrin complex withthiol-modulating properties. J. Med. Chem. 32:1426-1431—eachincorporated herein by reference in its entirety].

In one embodiment, a gold(III) complex having a diaminecyclohexaneligand and a ethylenediamine ligand is prepared with equimolar amountsof a gold(III) precursor salt, ethylenediamine and a diaminocyclohexane[i.e. cis-(1R,2S)-(1,2-diaminocyclohexane),cis-(1S,2R)-(1,2-diaminocyclohexane),trans-(1R,2R)-(−)-(1,2-diaminocyclohexane) ortrans-(1S,2S)-(+)-(1,2-diaminocyclohexane)gold(III) ethylenediamineincluding derivatives thereof]. Examples of the gold(III) salt includebut are not limited to hydrated or anhydrous salts of sodium gold(III)chloride, potassium gold(III) chloride, gold(III) chloride, gold(III)oxide, gold(III) hydroxide, gold(III) bromide and gold(III) sulfide. Thegold(III) precursor salt, the diaminocyclohexane and the ethylenediamineare dissolved separately in three portions of minimum ethanol orisopropyl alcohol at ambient temperature. Then, the gold(III) salt anddiaminocyclohexane solutions are mixed, preferably dropwise, stirredbriefly then filtered. The ethylenediamine solution is added to thefiltered mixture, preferably dropwise and thediaminocyclohexane-gold(III)-ethylenediamine solution is stirred for atleast overnight so that the final gold(III) complex product may beobtained as a white precipitate. The gold(III) complex product is washedrepeatedly and alternately with cold water, cold ethanol and thenfiltered and dried by heat or under reduced pressure in the presence ofa dessicating agent such as but not limited to phosphorus pentoxide andsilica gel.

Method of Inhibiting Proliferation of Cancer Cells and Inducing CancerCell Death

The present disclosure further provides a method of inhibitingproliferation of human cancer cells and inducing apoptosis of the humancancer cells in vitro or in vivo. Human cancer cells are contacted with1-100 μM of a gold(III) complex in accordance with the presentdisclosure or a composition comprising the gold(III) complex at thedefined concentration range, preferably 2-75 μM, more preferably 5-50μM, even more preferably 5-15 μM, 5-10 μM, 10-25 μM, 5-25 μM, 25-50 μMand 10-50 μM. The viability of cells can be determined by standard cellviability assays such as but not limited to ATP test, Calcein AM assay,clonogenic assay, ethidium homodimer assay, Evans blue assay,Fluorescein diacetate hydrolysis/propidium iodide staining assay, flowcytometry assay, formazan-based assays (MTT.XTT), green fluorescentprotein assay, lactate dehydrogenase assay, methyl vilet assay,propidium iodide assay, Resazurin assay. Trypan Blue assay and TUNELassay.

When contacted with one or more of the 1,2-(diaminocyclohexane)gold(III)ethylenediamine complexes at the defined concentration, the viability ofthe human cancer cells is reduced to at least 95%, preferably at least85%, more preferably at least 75%, even more preferably at least 50%, atleast 45%, at least 40%, at least 35%, at least 30%, at least 25%, atleast 20%, most preferably at least 15%, at least 12.5%, at least 10%,at least 7.5%, at least 5%, at least 2.5%, at least 2%, at least 1% andat least 0.5%.

The half maximal inhibitory concentration (IC₅₀) values of the gold(III)complexes against the human cancer cells are no higher than 100 μM,preferably at least no higher than 50 μM, more preferably no higher than30 μM, no higher than 20 μM, even more preferably no higher than 15 μM,no higher than 12 μM, most preferably no higher than 10 μM, no higherthan 5 μM and no higher than 2 μM. In some embodiments, the IC₅₀ valueof the gold (III) complexes against human prostate or gastric cancercells, such as but not limited PC3 and SGC7901 cell lines, are ranged1-20 μM, preferably 2-15 μM, more preferably 3-12 μM, even morepreferably 4-10 μM. In some embodiments, compared to cisplatin, the IC₅₀values of the gold(III) complexes provided herein are 0.5-5 times lower,preferably 0.5-3 times lower, more preferably 0.5-2 times lower, evenmore preferably 0.5-1 times lower.

In some embodiments, the human cancer cells are derived from commercialcell line models, including but are not limited to HeLa cervical cancercells, A549 lung cancer cells, HCT-15 colon cancer cells, HCT-8 or HRT-8colon cancer cells, DLD-1 colon cancer cells, MCF-7 breast cancer cells,A2780 ovarian cancer cells, A2780-cis cisplatin-resistant ovarian cancercells, PC3 prostatic cancer cells, DU-145 prostatic cancer cells,SGC7901 gastrointestinal cancer cells and SGC7901-ciscisplatin-resistant gastrointestinal cancer cells.

In other embodiments, the human cancer cells are cancer cells of a humanpatient who has been diagnosed with, is suspected of having, or issusceptible to or at risk of having at least one form of cancer,preferably prostate cancer and/or gastrointestinal cancer.

Methods of Treating Cancers and Combination Therapies

Cancers such as but not limited to sarcomas, carcinomas, melanomas,myelomas, gliomas and lymphomas can be treated or prevented with themixed diamine ligand gold(III) complexes provided herein. In someembodiments, methods incorporating the use of at least one of thegold(III) complexes of the present disclosure are effective in thetreatment or prevention of cancer of the blood, brain, bladder, lung,cervix, ovary, colon, rectum, pancreas, skin, prostate gland, stomach,breast, liver, spleen, kidney, head, neck, testicle, bone (includingbone marrow), thyroid gland or central nervous system. In someembodiments, these methods are especially effective in the treatment orprevention of cervical, colon and lung cancers.

The methods for treating cancer and other proliferative disordersdescribed herein inhibit, remove, eradicate, reduce, regress, diminish,arrest or stabilize a cancerous tumor, including at least one of thetumor growth, tumor cell viability, tumor cell division andproliferation, tumor metabolism, blood flow to the tumor and metastasisof the tumor. In some embodiments, after treatment with one or moregold(III) complexes or a pharmaceutical composition thereof, the size ofa tumor, whether by volume, weight or diameter, is reduced by at least5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 99% or 100%, relative to the tumor size before treatment. In otherembodiments, after treatment with the one or more gold(III) complexes ofa pharmaceutical composition thereof, the size of a tumor does notreduce but is maintained the same as the tumor size before treatment.Methods of assessing tumor size include but are not limited to CT Scan,MRI, DCE-MRI and PET Scan.

In some embodiments, the method for treating cancer and otherproliferative disorders involves the administration of a unit dosage ora therapeutically effective amount of one or more of gold(III) complexesor a pharmaceutical composition thereof to a mammalian subject(preferably a human subject) in need thereof. As used herein, “a subjectin need thereof” refers to a mammalian subject, preferably a humansubject, who has been diagnosed with, is suspected of having, issusceptible to, is genetically predisposed to or is at risk of having atleast one form of cancer. Routes or modes of administration are as setforth herein. The dosage and treatment duration are dependent on factorssuch as bioavailability of a drug, administration mode, toxicity of adrug, gender, age, lifestyle, body weight, the use of other drugs anddietary supplements, cancer stage, tolerance and resistance of the bodyto the administered drug, etc., then determined and adjustedaccordingly. The one or more of gold(III) complexes or a pharmaceuticalcomposition thereof may be administered in a single dose or multipleindividual divided doses. In some embodiments, the interval of timebetween the administration of gold(III) complexes or a pharmaceuticalcomposition thereof and the administration of one or more additionaltherapies may be about 1-5 minutes, 1-30 minutes, 30 minutes to 60minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12 hours, 12-24 hours, 1-2days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,15 weeks, 20 weeks, 26 weeks, 52 weeks, 11-15 weeks, 15-20 weeks, 20-30weeks, 30-40 weeks, 40-50 weeks, 1 month, 2 months, 3 months, 4 months 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 1 year, 2 years, or any period of time in between. In certainembodiments, mixed diamine ligand gold(III) compounds and one or moreadditional therapies are administered less than 1 day, 1 week, 2 weeks,3 weeks, 4 weeks, one month, 2 months, 3 months, 6 months, 1 year, 2years, or 5 years apart.

In certain embodiments, a gold(III) complex of the present disclosure ora pharmaceutical composition thereof may be used in combination with oneor more other antineoplastic or chemotherapeutic agents. A non-limitinglist of examples of chemotherapeutic agents are aflibercept,asparaginase, bleomycin, busulfan, carmustine, chlorambucil, cladribine,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, doxorubicin,etoposide, fludarabine, gemcitabine, hydroxyurea, idarubicin,ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan,mercaptopurine, methotrexate, mitomycin, mitoxantrone, pentostatin,procarbazine, 6-thioguanine, topotecan, vinblastine, vincristine,retinoic acid, oxaliplatin, cisplatin, carboplatin, 5-FU(5-fluorouracil), teniposide, amasacrine, docetaxel, paclitaxel,vinorelbine, bortezomib, clofarabine, capecitabine, actinomycin D,epirubicine, vindesine, methotrexate, tioguanine (6-thioguanine),tipifarnib. Examples for antineoplastic agents which are protein kinaseinhibitors include imatinib, erlotinib, sorafenib, sunitinib, dasatinib,nilotinib, lapatinib, gefitinib, temsirolimus, everolimus, rapamycine,bosutinib, pzopanib, axitinib, neratinib, vatalanib, pazopanib,midostaurin and enzastaurin. Examples for antineoplastic agents whichare antibodies comprise trastuzumab, cetuximab, panitumumab, rituximab,bevacizumab, mapatumumab, conatumumab, lexatumumab and the like.

EXAMPLES

The following examples have been included to further describe protocolsfor synthesizing and characterizing certain mixed diamine ligandgold(III) complexes (i.e. ethylenediamine and diaminocyclohexaneligands), and results thereof. It should be noted that these exampleshave been included for illustrative purposes, and are not intended tolimit the scope of the appended claims.

In the following examples, the synthesized gold(III) complexes 1-3containing ethylenediamine (en) and diaminocyclohexane (1,2-DACH) werecharacterized using elemental analyzer, solution and solid-state NMRmeasurements, UV-Vis, Mid- and Far-FTIR spectroscopic methods. The CHNanalysis data support the formation of the mixed en and 1,2-DACH ligandsgold(III) complexes 1-3 with general formula [(1,2-DACH)Au(en)]Cl₃. Thespectroscopic methods and NMR measurements confirm the formation ofgold(III) complexes containing bidentate en and 1,2-DACH ligands viaN-donor atoms. The computational studies corroborate spectroscopic dataof gold(III) complexes. The computational studies also demonstrate thattrans-(1,2-DACH)-gold(III)-(en) isomer is slightly more stable than thecis-(1,2-DACH)-gold(III)-(en) isomer. The coordination sphere of thesecomplexes around gold(III) center adopts distorted square planargeometry. According to antiproliferative effects of gold(III) complexes1-3 on prostate (PC3) and gastric (SCG7901) cancer cells, the order oftime dependent antiproliferative effect is complex 1 withcis-configuration>complex 3 with (1S,2S)(+)-configuration>complex 2 withtrans-configuration for both PC3 and SGC7901 cancer cells. Thecomparative studies lead to the conclusion that complex 1 withcis-configuration of 1,2-DACH may be the most promisingantiproliferative agent among mixed ligand based gold(III) complexes1-3. The inhibitory effect of complexes 1-3 on the proliferation ofrapidly dividing cells may be attributed to the induction of cell cycleblockage, interruption of the cell mitotic cycle, programmed cell death(apoptosis) or premature cell death (necrosis). The in vitrocytotoxicity results reveal that mixed diamine ligand gold(III)complexes are better anticancer agents than previously reported[Au(1,2-DACH)Cl₂]Cl, [Au(1,2-DACH)₂]Cl₃; and [Au(en)₂]Cl₃ and itsderivative complexes against gastric SGC7901 cancer cell line.

Moreover, gold(III) complexes 1 and 3 were more effective than[Au(1,2-DACH)Cl₂]Cl against prostate PC3 cancer cells. The followingexamples prove the huge potential of mixed diamine ligand gold(III)complexes in the treatment of human prostate and gastric cancers. Inparticular, [(cis-1,2-DACH)Au(en)]Cl₃ makes a strong candidate as apotential chemopreventative and chemotherapeutic agent against humangastric cancer.

Example 1 Chemicals, Cell Lines and Cell Cultures

Sodium tetrachloroaurate(III) dihydrate NaAuCl₄.2H₂O and ethylenediamine(en) were purchased from Sigma-Aldrich. cis-1,2-diaminocyclohexanecis-1,2-DACH (Formula X), trans-(±)-diaminocyclohexane trans-(±)-DACH(Formula Y), and (S,S)-(+)-diaminocyclohexane (S,S)-(+)-1,2-DACH(Formula Z) were purchased from Aldrich. Absolute C₂H₅OH, CH₃OH, D₂O andDMSO-d₆ were obtained from Fluka Chemicals Co. All other reagents aswell as solvents were obtained from Aldrich Chemical Co., and used asreceived.

Human gastric SGC7901 cancer and prostate PC3 cancer cell lines wereprovided by American Type Culture Collection (ATCC). Cells were culturedin Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetalcalf serum (FCS), penicillin (100 kU L⁻¹) and streptomycin (0.1 g L⁻¹)at 37° C. in a 5% CO₂-95% air atmosphere. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellowtetrazole) was purchased from Sigma Chemical Co, St. Louis, Mo., USA.

Example 2 Synthesis of Au(III) Complexes

Mixed ligand gold(III) chloride compounds namelycis-1,2-diaminocyclohexane ethylenediamine gold(III) chloride,[(en)Au{cis-(1,2-DACH)}]Cl₃ 1; trans-(±)-1,2-diaminocyclohexaneethylenediamine gold(III) chloride, [(en)Au{(trans-(±)-(1,2-DACH)}]Cl₃2; and (S,S)-(+)-1,2-diaminocyclohexane ethylenediamine gold(III)chloride [(en)Au{(S,S)-(+)-(1,2-DACH)}]Cl₃ 3; were synthesized by usingone mole equivalent of Sodium aurate dihydrate NaAuCl₄.2H₂O with onemole of ethylenediamine (en) and one mole equivalent of cis-(1,2-DACH)or (trans-(+)-(1,2-DACH) or (S,S)-(+)-(1,2-DACH) respectively accordingto modification of the synthesis in the literature [Al-Maythalony B A,Wazeer M I M, Isab A A (2009) Synthesis and characterization ofgold(III) complexes with alkyldiamine ligands. Inorg Chim Acta362:3109-3113; Al-Jaroudi S S, Fettouhi M, Wazeer M I M, Isab A A,Altuwaijri S (2013) Synthesis, characterization and cytotoxicity of newgold(III) complexes with 1,2-diaminocyclohexane: influence ofstereochemistry on antitumor activity. Polyhedron 50:434-442; Zhu S,Gorski W, Powell D R, Walmsley J A (2006) Synthesis, structures, andelectrochemistry of gold(III) ethylenediamine complexes and interactionswith guanosine 5′-monophosphate. Inorg Chem 45:2688-2694—eachincorporated herein by reference in its entirety].

Sodium tetrachloroaurate dihydrate NaAuCl₄.2H₂O, 398 mg (1.0 mmol) wasdissolved in minimum volume i.e. 10 mL of absolute ethanol at ambienttemperature. In a separate beaker, 1,2-diaminocyclohexane (1,2-DACH),114 mg (1.0 mmol) was dissolved in minimum volume i.e. 10 mL of absoluteethanol at ambient temperature. Both solutions were mixed dropwise andstirred for a half hour. Finally, a clear solution was obtained andfiltered. In a separate beaker, ethylenediamine (en), 120 mg (1.0 mmol)is dissolved in minimum volume i.e. 10 mL of absolute ethanol at ambienttemperature. The addition of (en) solution is added drop wise to theabove filtered solution. Upon stirring for overnight, the whiteprecipitate of [(en)Au(1,2-DACH)]Cl₃ was obtained. The product wasisolated, dissolved in 2 mL of water and filtered through Celite pad toremove NaCl. Addition of 100 mL of cold CH₃OH to the filtrate and awhite precipitate was obtained, filtered and washed with cold CH₃OH. Thesolid product was dried under reduced pressure with P₂O₅.

The yield of the compounds 1 (Formula 1a or 1b), 2 (Formula 2a or 2b)and 3 (Formula 3a or 3b) was in the range of 75-80%. Melting points andelemental analysis for complexes are presented in Table 1. The complexesprepared in the present study were characterized by FTIR and NMRmeasurements. The density functional calculations (DFC) studies basedhybrid B3LYP is also performed to analyze the structures of gold(III)complexes. All the data support the formation of the desired[(1,2-DACH)Au(en)]Cl₃ complexes.

TABLE 1 Melting point (MP) and CHN analysis of gold complexes 1, 2 and3. Found (calculated) % Complex MP (° C.) H C N (1) 161-163 6.57 22.2613.05 (6.64) (22.59) (13.17) (2) 175-178 6.59 22.32 13.01 (6.64) (22.59)(13.17) (3) 176-178 6.60 22.48 13.03 (6.64) (22.59) (13.17)

Example 3 Electronic Spectra

Electronic spectra where obtained for the gold(TIT) complexes usingLambda 200, Perkin-Elmer UV-Vis spectrometer. UV-Vis spectroscopy wasused to determine the stability of the complexes in a physiologicalbuffer (40 mM phosphate, 4 mM NaCl, pH 7.4). Electronic spectra wererecorded on freshly prepared of each complex in buffer solution at roomtemperature. Then, their electronic spectra were monitored over 7 daysat 37° C. The resulting UV-Vis absorption data are shown in Table 2.

TABLE 2 λ_(max) values derived from UV-Vis spectra for Au(III) complexes1, 2 and 3. Complex λ_(max) (nm) NaAuCl₄•2H₂O 293 (1) 335 (2) 338 (3)339

Example 4 Mid and Far-IR Studies

The solid-state FTIR spectra of the free ligands (1,2-DACH and en) andtheir corresponding mixed ligand gold(III) complexes were recorded on aPerkin-Elmer FTIR 180 spectrophotometer using KBr pellets over the range4,000-400 cm⁻¹. The selected mid-FTIR frequencies of free ligands andcorresponding mixed ligands gold(III) complexes are given in Table 3.Far infrared spectra were recorded for compounds 1, 2 and 3 at 4 cm⁻¹resolution at room temperature as cesium chloride (CsCl) disks on aNicolet 6700 FTIR with far-FTIR beam splitter. The selected far-FTIRdata for free ligands and their corresponding mixed ligand gold(III)complexes are given in Table 4. The references cited in Tables 3 and 4are Wadt W R, Hay P J (1985b) Ab initio effective core potentials formolecular calculations. Potentials for main group element Na to Bi. JChem Phys 82:284-298; Hartinger C G, Dyson P J (2009) Bioorganometallicchemistry—from teaching paradigms to medicinal applications. Chem SocRev 38:391-401; and Wadt W R, Hay P J (1985c) Ab initio effective corepotentials for molecular calculations. Potentials for K to Au includingthe outermost core orbitals. J Chem Phys 82:299-305, which areincorporated herein by reference in their entireties.

TABLE 3 Mid-FTIR frequencies, ν(cm⁻¹) for the mixed ligand Au(III)complexes 1, 2 and 3. Complex ν(N—H) ν_(shift) ν(C—N) ν_(shift) Refs. en3,393 w  1,033 m Wadt and Hay (1985b) [(en)AuCl₂]Cl 3,422 br 29 1,045 m12 Wadt and Hay (1985b) cis-(1,2-(DACH) 3,356 m, 3,286 m 1,092 s Hartinger and Dyson (2009) [{cis-(1,2-DACH)}AuCl₂]Cl 3,414 w  93 1,183s  91 Hartinger and Dyson (2009) (1) 3,395 br 74^(a), 2^(b) 1,182 w90^(a), 149^(b) This work trans-(±)-(1,2-DACH) 3,348 m, 3,271 m, 3,183 m1,082 m Hartinger and Dyson (2009) [{trans-(±)-(1,2-DACH)} 137, 149, 2011,175 m 93 Hartinger and Dyson (2009) AuCl₂]Cl (2) 3,432 br 168^(a),39^(b) 1,180 m 93^(a), 147^(b) This work (S,S)-(+)-(1,2-DACH) 3,340 m,3,252 m, 3,167 m 1,082 m Hartinger and Dyson (2009) [{S,S-(+)- 1,171 m89 Hartinger and Dyson (2009) (1,2-DACH)}AuCl₂]Cl (3) 3,386 br 132^(a),−7^(b) 1,180 m 98^(a), 147^(b) This work ^(a)With respect to (DACH)^(b)With respect to (en)

TABLE 4 Far-FTIR frequencies, ν(cm⁻¹) for the mixed ligand Au(III)complexes 1, 2 and 3. Complex Au—Cl Au—N Refs. NaAuCl₄•2H₂O 365 — Thiswork [(en)AuCl₂]Cl — 391, 474 Wadt and Hay (1985c) [cis-(1,2- 352, 367437 Hartinger and Dyson (2009) DACH)AuCl₂]Cl (1) — 326, 417 This work[trans-(±)-(1,2- 353, 365 437 Hartinger and Dyson (2009) DACH)AuCl₂]Cl(2) — 391, 442 This work [(S,S)-(+)-(1,2- 353, 366 395, 436 Hartingerand Dyson (2009) DACH)AuCl₂]Cl (3) — 376, 440 This work

Example 5 Solution NMR Measurements

All NMR measurements were carried out on a Jeol JNM-LA 500 NMRspectrophotometer at 298 K. The ¹H NMR spectra were recorded at afrequency of 500.00 MHz. The ¹³C NMR spectra were obtained at afrequency of 125.65 MHz with ¹H broadband decoupling. The spectralconditions were: 32 k data points, 0.967 s acquisition time, 1.00 spulse delay and 45 pulse angle. The chemical shifts are referenced to1,4-dioxane as an internal standard in 13C NMR measurements. The ¹H and¹³C NMR spectra chemical shifts are given in Tables 5 and 6, accordingto Formulas 1a-b, 2a-b and 3a-b.

TABLE 5 ¹H NMR chemical shifts of free ligands and complexes 1, 2 and 3in D₂O. ¹H (δ in ppm) H3, H6 H3, H6 H4, H5 H4, H5 Compound H1, H2 (eq)(ax) (eq) (ax) H1′, H2′ Refs. en — — — — — 3.2, s This workcis-(1,2-DACH) 2.23, m 1.85, m 1.69, m 1.28, m 1.12, m — Hartinger andDyson (2009) (1) 3.61, m 1.96, m 1.77, m 1.59, m 1.41, m 3.16, s Thiswork trans-(±)-(1,2-DACH) 2.25, m 1.85, m 1.68, m 1.28, m 1.11, m —Hartinger and Dyson (2009) (2) 3.05, m 2.11, m 1.54, m 1.48, m 1.10, m3.14, s This work (S,S)-(+)-(1,2-DACH) 2.24, m 1.85, m 1.69, m 1.28, m1.11, m — Hartinger and Dyson (2009) (3) 3.08, m 2.19, m 1.63, m 1.54, m1.19, m 3.17, s This work

TABLE 6 Solution-state¹³C NMR chemical shifts of free ligands andcomplexes 1, 2 and 3 in D₂O. ¹³C (δ in ppm) Compound C1, C2 C3, C6 C4,C5 C1′, C2′ en — — — 37.67 cis-(1,2-DACH) 58.2 35.26 26.36 — (1) 61.7426.13 20.64 50.39 trans-(±)-(1,2-DACH) 58.46 35.55 26.63 — (2) 64.5932.95 24.12 50.63 (S,S)-(+)-(1,2-DACH) 58.27 35.32 26.43 — (3) 64.4432.84 24.02 50.48

Example 6 Solid State NMR Studies

Solid-state ¹³C NMR spectra were recorded on a Bruker 400MHzspectrometer at ambient temperature of 298 K. Samples were packedinto 4 mm zirconium oxide (ZrO) rotors. Cross polarization (CP) and highpower (HP) decoupling were employed. Pulse delay of 7.0 s and a contacttime of 5.0 ms were used in the CPMAS experiments. The magic anglespinning (MAS) rates were maintained at 4 and 8 kHz. 13C chemical shiftswere referenced to tetramethylsilane (TMS) by setting the high frequencyisotropic peak of solid adamantane to 38.56 ppm. The solid-state NMRdata are given in Table 7.

TABLE 7 Solid-state¹³C NMR chemical shifts of free ligands and complexes1, 2 and 3 in D₂O. ¹³C (δ in ppm) Complex C1, C2 C3, C6, C4, C5 C1′, C2′Refs. [cis-(1,2-DACH)}AuCl₂]Cl 66.20, 65.35 30.98 27.02, 22.12 —Hartinger and Dyson 2009) (1) 64.32 28.85 28.85, 22.94 54.3  This work[trans-(±)-(1,2-DACH)AuCl₂]Cl 69.6  37.37 27.99 — Hartinger and Dyson(2009) (2) 69.60, 65.45 36.63 27.53 54.08 This work[(S,S)-(+)-(1,2-DACH)AuCl₂]Cl 70.21 37.86 29.16 — Hartinger and Dyson(2009) (3) 67.1  36.19 27.65 54.18 This work

Example 7 Stability Determination of the Gold(TIT) Complexes

The stability of complexes 1, 2 and 3 were tested in water aswell as ina mixture of solvents i.e. DMSO/water (2/1 in v/v ratio) by ¹H and ¹³CNMR measurements. To investigate the structural stability of thecomplexes, NMR spectra of the complexes dissolved in D₂O; and in mixedDMSO-d₆/D₂O (2/1 in v/v ratio) solution were obtained just afterdissolution, 24 h and 1 week at room temperature in mixed DMSO-d₆/D₂Oand at 37° C. in D2O. At least 20 mg of complexes 1, 2 and 3 in 1 mL D₂Oat 37° C.; and in 1 mL DMSO-d₆/D₂O (2/1:v/v) at room temperature weresubjected to ¹H and ¹³C NMR measurements and followed by their spectralanalysis. Immediately after dissolution of complexes 1, 2 and 3 in therespective solvents and duplicate samples were then stored at roomtemperature and 37° C., respectively, and analyzed again after 24 h and1 week in order to determine stability of complexes. Since the complexeswere not used beyond a week after dissolution, so NMR measurements werelimited to one week.

Example 8 Electrochemistry

The electrochemical experiments were performed at room temperature usinga potentiostat (SP-300, Bio-Logic Science Instruments) controlled byEC-Lab v10.34 software package. The electrochemical experiments wereperformed at room temperature. All the measurements were performed onsolutions de-aerated by bubbling ultra-pure nitrogen for 15 min. Thevalues of reduction potential here reported were measured against asaturated calomel electrode (SCE). The cyclic voltammetry of thecompounds 1, 2 and 3 were measured at scan rate of 50 mV/s on areference buffer (40 mM phosphate, 4 mM NaCl, pH 7.4) using platinum asworking electrode and graphite as a counter electrode with aconcentration of 1.0 mM at room temperature. Ferrocene was used aspseudo reference to calibrate the working electrode. The couple Fe/IIVIIformal potential of ferrocene occur at E^(o/)=+0.44 V (vs SCE) in 0.1MBu₄NPF₆ solution in CH₃CN solvent which is similar to the report valueunder the same experimental condition [Hans J, Beckmann A, Kruger H-J(1999) Stabilization of Copper(III) ions with deprotonatedhydroxyiminoamide ligands: syntheses, structures, and electronicproperties of Copper(II) and Copper(III) complexes. Eur J Inorg Chem1:163-172—incorporated herein by reference in its entirety]. Conversionto values vs ENH was obtained upon adding +0.24 V to the correspondingSCE values.

Example 9 Computational Studies

The structures of the [(1,2-DACH)Au(en)]³⁺ complexes in their fourpossible conformations (cis-S,R or 1(a) as in Table 8; cis-R,S or 1(b);trans-S,S or 2(a), and trans-R,R or 2(b)) were studied without anygeometrical constrains using GAUSSIAN09 program [Frisch M J, Trucks G W,Schlegel H B, Scuseria G E, Robb M A, Cheeseman J R, Scalmani G, BaroneV, Mennucci B, Petersson G A, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov A F, Bloino J, Zheng G, Sonnenberg J L, Hada M, Ehara M,Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O,Nakai H, Vreven T, Montgomery J A, Jr., Peralta J E, Ogliaro F, BearparkM, Heyd J J, Brothers E, Kudin K N, Staroverov V N, Kobayashi R, NormandJ, Raghavachari K, Rendell A, Burant J C, lyengar S S, Tomasi J, CossiM, Rega N, Millam J M, Klene M, Knox J E, Cross J B, Bakken V, Adamo C,Jaramillo J, Gomperts R, Stratmann R E, Yazyev O, Austin A J, Cammi R,Pomelli C, Ochterski J W, Martin R L, Morokuma K, Zakrzewski V G, Voth GA, Salvador P, Dannenberg J J, Dapprich S, Daniels A D, Farkas O”,Foresman J B, Ortiz J V, Cioslowski J, and Fox D J (2009) Gaussian 09,Revision A. 1, Gaussian, Inc., Wallingford Conn.—incorporated herein byreference in its entirety]. The hybrid B3LYP density functional (thethree-parameter Becke functional with correlation fromthe Lee-Yang-Parrfunctional) with the Los Alamos National Laboratory-2 double-ζ (LANL2DZ)basis set was employed in this study [Becke A D (1988)Density-functional exchange-energy approximation with correct asymptoticbehavior. Phys Rev 38:3098; Wadt W R, Hay P J (1985a) Ab initioeffective core potentials for molecular calculations. Potentials for thetransition metal atoms Sc to Hg. J Chem Phys 82:270-283; Wadt W R, Hay PJ (1985b) Ab initio effective core potentials for molecularcalculations. Potentials for main group element Na to Bi. J Chem Phys82:284-298; Wadt W R, Hay P J (1985c) Ab initio effective corepotentials for molecular calculations. Potentials for K to Au includingthe outermost core orbitals. J Chem Phys 82:299-305—each incorporatedherein by reference in its entirety]. Previously reported results forsome gold(III)-based complexes at this level of calculations areconsistent with the experimental findings described herein[Al-Maythalony B A, Wazeer M I M, Isab A A (2009) Synthesis andcharacterization of gold(III) complexes with alkyldiamine ligands. InorgChim Acta 362:3109-3113—incorporated herein by reference in itsentirety]. Moreover, the stationary points have been confirmed byfrequency calculation. Selected bond lengths and bond angles are givenin Table 8 for the four molecular conformations, while Table 9 comparesthe relative stabilities based on the calculated energies of theseminimum structures.

TABLE 8 Selected bond lengths and bond angles of the[(1,2-DACH)Au(en)]³⁺ complex in its four possible conformations. 1(a)1(b) 2(a) 2(b) Bond lengths (Å) Au—N₁ 2.147 2.14 2.145 2.146 Au—N₂ 2.1472.132 2.145 2.146 Au—N₃ 2.131 2.118 2.134 2.135 Au—N₄ 2.132 2.127 2.1342.135 N₁—C 1.538 1.54 1.539 1.539 N₂—C 1.538 1.541 1.539 1.539 N₃—C1.544 1.559 1.545 1.544 N₄—C 1.545 1.552 1.545 1.544 Bond angles (°)N₁—Au—N₃ 177.7 179.5 179.4 179.5 N₂—Au—N₄ 177.6 179.4 179.4 179.5N₁—Au—N₂ 81.8 82.1 82 81.9 N₂—Au—N₃ 100.5 98.3 98.6 98.6 N₃—Au—N₄ 77.281.7 80.9 80.9 N₄—Au—N₁ 100.6 97.9 98.5 98.6 Au—N₁—C 109.3 109.1 109 109Au—N₂—C 109.1 109.1 109 109 Au—N₃—C 111.4 109.5 110.7 110.8 Au—N₄—C111.8 109.4 110.7 110.8

TABLE 9 Relative energies of the four possible conformations of thecomplexes 1 and 2. Conformation Relative energy (kcal/mol) 1(a) 3.611(b) 3.1 2(a) 0 2(b) 0.11

Example 10 MTT Assay for Antiproliferative Effects of[(1,2-DACH)Au(En)]Cl₃ Compounds 1, 2 and 3 on PC3 and SGC7901 CancerCells

An MTT assay was used to obtain the number of living cells in thesample. Human gastric cancer SGC7901 and prostate cancer PC3 cells wereseeded on 96-well plates at a predetermined optimal cell density, i.e.ca. 6,000 cells/100 μL per well in 96-well plates, to ensure exponentialgrowth in the duration of the assay. After 24 h pre-incubation, thegrowth medium was replaced with the experimental medium containing theappropriate drug, using one of gold(III) compounds 1, 2 and 3 or acontrol using water. Six duplicate wells were set up for each sample,and cells untreated with drug served as a control. In one set of cultureplates, human gastric cancer SGC7901 and human prostate PC3 cells weretreated with 10 μM compounds 1, 2 and 3 as the drug and the control(water) for 24, 48 and 72 h. In other sets, the compounds 1, 2 and 3with different concentration, i.e. 10, 20 and 30 μM, were employed todetermine the growth inhibitory effect for both PC3 and SGC7901 cellsseparately. After incubation, 10 μL MTT (6 g/L, Sigma) was added to eachwell and the incubation was continued for 4 h at 37° C. After removal ofthe medium, MTT stabilization solution [dimethylsulfoxide (DMSO):ethanol (C₂HSOH)=1:1 in v/v ratio] was added to each well, and shakenfor 10 min until all crystals were dissolved. Then, the optical densitywas detected in a micro plate reader at 550 nm wavelength using anEnzyme-Linked Immuno-Sorbent Assay (ELISA) reader. After being treatedwith gold(III) complexes 1, 2 and 3, the cell viability was examined byMTT assay. Each assay was performed in triplicate. An MTT assay for theinhibitory effect has been used for compounds 1, 2 and 3 against PC3 andSGC7901 cells. These cells were treated with various concentrations ofcompounds 1, 2 and 3 for 24-72 h. All results are shown in FIGS. 1-7Aand B.

Example 11 In Vitro Cytotoxic Assay for PC3 and SGC7901 Cancer Cells

Human prostate PC3 and gastric SGC7901 cells were used throughout theexamples of the present disclosure. Cells were cultured in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% fetal calf serum(FCS), penicillin (100 kU L⁻¹) and streptomycin (0.1 g L⁻¹) at 37° C. ina 5% CO2-95% air atmosphere. Human gastric SGC7901 cells and prostatePC3 were incubated with these compounds at fixed concentrations or withwater as a control to assess the inhibitory effect on cell growth. Thestandard MTT assay has been used to assess the inhibitory effect on cellgrowth. The cell survival versus drug concentration is plotted.Cytotoxicity was evaluated in vitro with reference to the IC₅₀ value.The half maximal inhibitory concentration (IC₅₀) is a measure of theeffectiveness of a compound to inhibit biological or biochemicalfunctions. According to the FDA, ICo₅₀ represents the concentration of adrug/compound/complex that is required for 50% inhibition in vitro. Itis evaluated from the survival curves as the concentration needed for a50% reduction of survival. ICo₅₀ values are expressed in μM. The IC₅₀values were calculated from dose-response curves obtained in replicateexperiments. The IC₅₀ data are presented in Table 10.

TABLE 10 In vitro cytotoxicity data of the complexes 1, 2 and 3 afterexposure of 72 h towards human cancer SGC1901 and PC3 cell lines. IC₅₀(μM)^(a) Complex SGC7901 PC3 Cisplatin 7.3 ± 0.5 1.1 ± 0.1 (1) 5.5 ± 0.24.8 ± 0.1 (2) 7.9 ± 0.2 8.9 ± 0.1 (3) 5.8 ± 0.2 6.1 ± 0.1

Example 12 UV-Vis Spectra

The λ_(max) values obtained from UV-Vis spectra for the complexesstudied are shown in Table 2. The gold(III) compounds 1, 2 and 3exhibit, in a reference buffered phosphate solution, intense absorptionsin the range 335-339 nm, which are assigned as ligand-to-metalcharge-transfer (LMCT) transitions characteristically associated to thegold(III) center. These absorption bands were previously assigned toNH—Au(III) charge-transfer bands [Kimura E, Kurogi Y, Takahashi T (1991)The first gold(III) macrocyclic polyamine complexes and application toselective gold(IIT) uptake. Inorg Chem 30:4117-4121—incorporated hereinby reference in its entirety]. It is worth-mentioning that thesespectral features appear only at relatively high pH values (pH>6-7) atwhich the deprotonation of ligand has fully occurred. According tocrystal field theory for d⁸ complexes the lowest unoccupied molecularorbital (LUMO) orbital is d_(x2-y2), so ligand to metal charge transfercould be due to p_(σ)→d_(x2-y2) transition [Haruko I, Junnosuke F, KazuoS (1967) Absorption spectra and circular dichroisms of metal complexes.I. Platinum(II)-, palladium(II)- and gold(III)-complexes containingoptically active diamines. Bull Chem Soc Jpn 40:2584-2591—incorporatedherein by reference in its entirety].

The electronic spectra of compounds 1, 2 and 3 were monitored at 37° C.over 3 days after mixing in the buffer solution. The electronic spectrafor compounds 1, 2 and 3 at just after mixing; and after 3 days areillustrated in FIGS. 8A-8C. It is apparently observed that thetransitions remain relatively unmodified over a period of 3 days. Suchobservations show a substantial evidence for the stability of thesecompounds 1, 2 and 3 under the conditions of solution state.Nevertheless, a slight decrease in intensity of the characteristic bandswas noticed with time without significant modifications in shape ofspectra. Further, such observation indicates that the gold center inthese compounds remains in the +3 oxidation state. The minor spectralchanges that are generally observed within the first hours may beascribed either to dissociation of the amine ligands from the gold(III)complex or to partial reduction of gold(III) to metallic gold. Ingeneral, however, loss of spectral intensity is lower than 10% of theoriginal intensity within the observation period of 7 days whichindicates high stability of these compounds in the buffer. It is apossible proposition that compounds 1, 2 and 3 would be stable enough inthe physiological environment to undergo the necessaryreactions/interactions required for bioactivity, without decomposition.

Example 13 Mid- and Far-FTIR Spectroscopic Characterization

The most significant bands recorded in the FTIR spectra of the ligand,[(1,2-DACH)Au(en)]Cl₃ complexes have been reported in Tables 3 and 4. Itis noted that N—H stretching vibrations of complexes 1, 2 and 3 exhibit,in the range 3,386-3,432 cm-1, blue shifting compared with the aminogroup of the corresponding free ligands. This is most likely due tostronger H-bonding interactions in the free ligands as compared to twocoordinated amino-:NH2 groups of 1,2-diaminocyclohexane (1,2-DACH) viadonor N atoms, leading to formation of five member chelate withgold(III) center in corresponding compounds 1, 2 and 3. The coordinationof amino-:NH2 with Au(III) center via nitrogen donor atom and formationof Au—N bond can be supported by the presence of a m(Au—N) band at417-442 cm-1 in the Far-FTIR [Beck W, Fehlhammer W P, Pollmann P,Schuierer E, Feldl K (1967) Darstellung, IR-und Elektronenspektren vonAzido-Metall-Komplexen. Chem Ber 100:2335-2361—incorporated herein byreference in its entirety]. The C—N stretching bands also showed asignificant shift to higher wave number, indicating a shorter C—N bondin the compound than in the free ligand. Moreover, there was no signalobserved at 352 and 367 cm-1 corresponding to the symmetric andasymmetric stretching of the Cl—Au—Cl bonds in [(1,2-DACH)AuCl₂]⁺ typecompounds, indicating the absence of the mono-(1,2-DACH)gold(III)chloride compound [Al-Maythalony B A, Wazeer M I M, Isab A A (2009)Synthesis and characterization of gold(III) complexes with alkyldiamineligands. Inorg Chim Acta 362:3109-3113—incorporated herein by referencein its entirety]. The [(1,2-DACH)Au(en)]Cl₃ complexes 1, 2 and 3 showN—H stretching frequencies generally lower in comparison with[(1,2-DACH)AuCl₂]Cl complexes (Table 3), most probably due to strongerhydrogen bonding interactions with the chloride anions in the[(DACH)Au(en)]Cl₃ complexes. Furthermore the Au—N stretching frequenciesare consistent with weaker Au—N bond strength in complexes 1, 2 and 3compared to [(1,2-DACH)AuCl₂]Cl complexes.

Example 14 Solution-State NMR Characterization

All ¹H NMR spectra supported the structures of the synthesized complexesas indicated by the integration of the signals of C—H protons connectedto the amino groups of the (1,2-DACH) and (en). For example, the ratioof the protons attached to amino group in both (1,2-DACH) and (en) forcomplex 3 is 1:2 as illustrated in FIG. 9. Its ¹³C NMR spectrum is alsoconfirmed the complex structure as shown in FIG. 10. The ¹H and ¹³C NMRchemical shifts of compounds 1-3 along with their corresponding freeligands are listed in Tables 5 and 6, respectively. In the ¹H and ¹³CNMR spectra of complexes 1, 2 and 3, one half of the total number ofsignals were noticed because of the C₂ symmetry of the1,2-diaminocyclohexane ring, which is considered as a rigid conformerthat allowed, for instance, to distinguish equatorial H3 and H6 fromaxial H3 and H6 at room temperature. The signals of C—H protonsconnected to the amino groups for both (1,2-DACH) and (en) occur at3.05-3.61 ppm, shifting downfield compared with the correspondingsignals at 2.23-2.65 ppm in the free diamine ligands. The significantdownfield shift was observed at 3.62 ppm for complex 1 with respect tothe free cis-1,2-DACH ligand at 2.23 ppm. This can be attributed to thedonation of nitrogen lone pairs to the gold center that causesde-shielding of the proton(s) next to the bonding nitrogen. On the otherhand, ¹³C NMR downfield shift was observed only for the carbon next tothe bonding nitrogen and the others carbons in the complex for(1,2-DACH) showed upfield shift presumably due to γ-shielding effect.For instance, chemical shift of C3 and C4 for complex 1 observed at26.13 and 20.64 ppm, respectively, whereas, for free diamine ligand itoccurs at 35.26 and 26.36 ppm. It is also worth-mentioning thatcomplexes 1-3, even though they have the same skeleton of (1,2-DACH) and(en), their carbon chemical shifts were not the same due to a differentstereochemistry upon coordination.

Example 15 Solid-State NMR Characterization

As listed in Table 7, solid state NMR spectrum of complex 3 showedequivalency in the chemical shifts of carbon atoms (C1, C2), (C3, C6),(C4, C6) and (C1, C2) where two sets of peaks were observed, whereas, asimilar behavior was not observed for carbon atoms of (DACH) incomplexes 1 and 3. This indicates that these complexes 1 and 3 in thesolid state lack C₂ symmetry, due to packing effect. In contrast, allsynthesized complexes 1, 2 and 3 showed C₂ symmetry in the solutionstate as indicated earlier by solution ¹H and ¹³C NMR.

Compared to solution chemical shifts, significant de-shielding in solidstate is observed with similarity in chemical shift trends among allcomplexes 1-3 as given in Table 7, which is a clear indication ofstability of the structural similarity in solid state as well as insolution state.

Example 16 Computational Analysis

The structures of the [(1,2-DACH) Au(en)]³⁺ complexes as obtained fromthe B3LYP/LANL2DZ level of calculations are shown in FIGS. 11A-D.Selected quantitative structural parameters are also listed in Table 8.The complexes show a distorted square planar geometry structure aroundthe gold(III) center. The N—Au—N angles in most of the conformations arewithin less than a degree from the perfect square planar geometry. TheAu—N was predicted to be in the range of 2.12-2.15 Å for both (1,2-DACH)and (en) bidentate diamine ligands. The C—N bond length shows asignificant increase by ca. 0.1 Å when it is compared with the same typeof bonds in normal amines [Allen F H, Kennard O, Watson D G, Brammer L,Orpen A G (1987) Tables of bond lengths determined by X-ray and neutrondiffraction. Part 1. bond lengths in organic compounds. J Chem SocPerkin Trans II:S1-S19—incorporated herein by reference in itsentirety].

The four coordinated nitrogen atoms (two N from 1,2-DACH and two N fromen) are predicted to adopt a sp³ type of hybridization as it can easilybe concluded by viewing the calculated bond angles (Table 8). From thecomputed energetics of the four structures of the complexes 1 and 2(Table 9), the trans-conformations are more preferable compared to thecis-conformations with more than 3.5 kcal/mol difference. The mostpossible explanation of this energy variation is the ring configurationof the 1,2-DACH ligand, where the methylene (CH₂) units experience moresteric repulsion in the cis form in comparison to that in the transform.

Example 16 Stability Determination of Mixed Diamine Ligand Gold(III)Compounds

NMR spectra of the complexes were obtained upon immediate dissolution toserve as reference spectra and later at 24 h and after 7 days at 37° C.in D₂O and at room temperature in mixed DMSO-d₆/D₂O in order todetermine their stability. In general, all complexes showed highstability in D₂O as well as in mixed DMSO-d₆/D₂O and their NMR profilesremained unchanged over the span of 7 days. For example, FIGS. 12A-B and13A-B illustrated, respectively, the ‘H and’³C NMR profiles of thecompound 1 at just after mixing and after 7 days. Whereas, thesecompounds in mixed DMSO-d₆/D₂O solvent system were slightly less stableat the experimental conditions, in which, minor dissociation ofethylenediamine (en) out of the gold complexes was observed in 24 h. Onthe other hand, no dissociation was observed for (1,2-DACH). Among allsynthesized complexes, the maximum dissociation for ethylenediamine (en)after 7 days was experienced for compound 3 with 25%. ¹H and ¹³C NMRprofiles of compound 3 in DMSO-dd/D₂O at just after mixing and after 7days as shown in FIGS. 14A-B and 15A-B respectively. ¹H and ¹³C NMR ofcompound 3 spectra after 7 days in DMSO-d6/D2O showed extra peak at 3.07and 37.24 ppm as shown in FIGS. 14B and 15B, respectively, correspondingto the free (en) atoms. It is concluded that the bond between gold(III)and (1,2-DACH) is stronger than the bond between gold(III) and (en) inthese complexes 1-3, suggesting that ethylenediamine (en) could be abetter leaving group.

Example 17 Electrochemical Behavior of Mixed Diamine Ligand Gold(III)Complexes

The electrochemical behavior of compounds (1, 2 and 3 was investigatedin a physiological environment through cyclic voltammetry (CV). Thecyclic voltammetric curves of the complexes 1, 2 and 3 are shown inFIGS. 16A-C. Table 11 summarizes the cyclic voltammetric data of all thestudied compounds. The reduction potential values vs. NHE for thereduction processes exhibited by the complexes 1, 2 and 3, in areference phosphate buffer solution, were in the range (+0.46)-(+0.51)V. Cyclic voltammetric data indicated that trans-1,2-DACH conformer isslightly more stable than the cis-1,2-DACH conformer of the complexeswhich is also corroborated by UV-Visible spectral studies. Gold(III)complexes 1, 2 and 3 show one irreversible reduction process in whichthe controlled potential coulometry involves three electrons per mole.The occurrence of Au(III)/Au(0) reduction is visually indicated by theappearance of a thin gold layer deposited on the platinum electrodesurface after exhaustive electrolysis (Ew, −0.7 V). In general, cyclicvoltammetric results suggest that these compounds are quite stable underthe physiological conditions.

The stability of the gold(III) compounds in the reference phosphatebuffer was also checked after the addition of stoichiometric amounts ofthe biologically important reducing agent sodium ascorbate. It wasobserved that all complexes were quickly and almost completely reducedin 60 min.

TABLE 11 Peak potential values vs. ENH for reduction of gold(III)complexes. Complex E_(p) (V) (1) 0.49 (2) 0.46 (3) 0.51

Example 18 Antiproliferative Effects of Gold(III) Complexes 1, 2 and 3on Prostate (PC3) and Gastric (SGC7901) Cancer Cells

Modern oncologic or anticancer studies aim towards designing newercompounds showing enhanced antiproliferative potential and not as muchof associated toxicity than cisplatin. In this connection, gold(III)complexes with various ligands including Au—N, Au—S or Au—C bonds arebeing extensively developed and investigated for their bioactivities asantiproliferative agents [Ott I, Gust R (2007) Non platinum metalcomplexes as anticancer drugs. Arch Pharm Chem Life Sci 340:117-126;Ahmed A, Al Tamimi D M, Isab A A, Alkhawajah A M M, Shawarby M A (2012)Histological changes in kidney and liver of rats due to gold(III)compound [Au(en)Cl₂]Cl. PLoS ONE 7:e51889—each incorporated herein byreference in its entirety]. In the present disclosure, a new series ofgold(III) complexes 1-3 containing mixed ethylenediamine (en) and1,2-DACH ligands are being evaluated for antiproliferation against PC3and SGC7901 cancer cell lines. FIGS. 1-3A and B illustrated timedependent antiproliferative effects of complexes 1, 2 and 3respectively. In the time dependent, the growth inhibition on PC3 andSGC7901 cancer cells was studied using fixed concentration i.e. 10 μM.It can be seen from these figures that time-dependent antiproliferativeeffects of complexes 1, 2 and 3 on PC3 cancer cells are better thanthose on SGC7901 cancer cells. Complexes 1 and 3 showed better cellinhibition against PC3 cell line than complex 2 as shown in thesefigures. However, Complex 1 showed better cell inhibition againstSGC7901 cancer cell line than complexes 2 and 3 as shown in FIGS. 1-3Aand B. Gold(III) complexes 1 and 3 demonstrated a comparable cellinhibition; against PC3 cell line as shown in FIGS. 1A and B, 3A and B,whether the complexes exposure time was 24 or 72 h. All the gold(III)complexes showed lower cell inhibition against both cancer cell linesfor 72 h exposure time compared to 24 h as shown in FIGS. 1-3A and B.

Cell growth inhibition is also dependent on concentration of the drug.So, concentration-dependent cell growth inhibition studies of gold(III)complexes 1-3 against human prostate PC3 and gastric SGC7901 cancercells were conducted by using 10 and 20 μM concentrations. The resultsincluded cell inhibition augmented with the increase in concentration ofthe complexes 1, 2 and 3 as shown in FIGS. 4-6A and B, respectively. Itis generally observed from these figures that concentration-dependentantiproliferative effects of complexes 1, 2 and 3 on PC3 cancer cellsare superior to those on SGC7901 cancer cells. In theconcentration-dependent cell growth inhibition study at twoconcentrations (10 and 20 μM), complex 1 showed better cell inhibitionagainst SGC7901 cancer cell line than complexes 2 and 3 as shown inFIGS. 4-6A and B; whereas complexes 1 and 3 showed better cellinhibition against PC3 cell line than complex 2, as shown in thefigures. Gold(III) complexes 1 and 3 demonstrated a comparable cellinhibition; against PC3 cell line at 20 μM concentration as shown inFIGS. 4A and B and 6A and B, respectively.

FIGS. 7A and 7B compare the time-dependent antiproliferative effects of10 μM complexes 1, 2 and 3 on both PC3 and SGC7901 cancer cells for 24and 72 h. It has been observed that the order of time dependentantiproliferative effect is complex 1>complex 3>complex 2 for both PC3and SGC7901 cancer cells. Such comparative study leads to the conclusionthat complex 1 may be the most effective antiproliferative agent amongmixed ligand based gold(III) complexes 1-3.

The exact mechanisms on antiproliferation of [(DACH)Au(en)]Cl₃-typecomplexes on PC3 and SGC 7901 cancer cell lines remain unclear. Thesignificantly diminished renal toxicity of ethylenediamine complex ofgold(III) could be attributed to their different antiproliferativemechanism of action and selective sparing of the proximal tubularepithelial cells [Ahmed A, Al Tamimi D M, Isab A A, Alkhawajah A M M,Shawarby M A (2012) Histological changes in kidney and liver of rats dueto gold(III) compound [Au(en)Cl₂]Cl. PLoS ONE 7:e51889—incorporatedherein by reference in its entirety].

Most gold(III) compounds display reduced affinity for DNA and it seemsreasonable that DNA is neither the primary nor the exclusive target formost gold(III) complexes. Recent studies have proposed a different modeof action for these compounds, in most of the cases, induce apoptosiswas the mode of cell death [Vivek S, Kyoungweon P, Mohan S (2009)Colloidal dispersion of gold nanorods: Historical background, opticalproperties, seed-mediated synthesis, shape separation and self assembly.Mater Sci Eng R 65:1-38; Niemeyer C M (2001) Nanoparticles, proteins,and nucleic acids: biotechnology meets materials science. Angew ChemIntl Ed 40:4128-4158; Pellegrino T, Kudera S, Liedl T, Javier A M, MannaL, Parak W J (2005) On the development of colloidal nanoparticlestowards multifunctional structures and their possible use for biologicalapplications. Small 1:48-63—each incorporated herein by reference in itsentirety]. Their mechanism however, is not precisely delineated.However, the mechanisms associated with the inhibitory effects ofcomplexes 1-3 on the proliferation of rapidly dividing cancer cells maybe comprised of a cumulative impact on the induction of cell cycleblockage, interruption of the cell mitotic cycle, apoptosis (programmedcell death) and necrosis (premature cell death) [Taatjes D J, Sobel B E,Budd R C (2008) Morphological and cytochemical determination of celldeath by apoptosis. Histochem Cell Biol 129:33-43; Takemura G,Minatoguchi M S, Fujiwara H (2013) Cardiomyocyte apoptosis in thefailing heart—a critical review from definition and classification ofcell death. Int J Cardio 167:2373-2386; Hayashi R, Nakatsui K, SugiyamaD, Kitajima T, Oohara N, Sugiya M, Osada S, Kodama H (2014) Anti-tumoractivities of Au(I) complexed with bisphosphines in HL-60 cells. J InorgBiochem 137:109-114—each incorporated herein by reference in itsentirety].

Example 19 In Vitro Cytotoxicity of Gold(III Complexes 1-3 on Prostate(PC3) and Gastric (SGC7901) Cancer Cells

Milovanovic et al. have studied the cytotoxicity studies of [Au(en)Cl₂]+and [Au(SMC)Cl₂]⁺ where SMC=S-methyl-_(L)-cysteine and [Au(DMSO)₂Cl₂]⁺(DMSO=dimethyl sulphoxide). They concluded that gold(III) complexes aremuch faster to react with nucleophiles compare to Pt(II) complexes. Theyalso demonstrated that gold(III) complexes exhibit relevant cytotoxicproperties when tested on chronic lymphocytic leukemia cells (CLL). Thisconclusion indicates that gold(III) complexes have good potential forthe treatment of cancer. In addition [Au(en)Cl₂] complex showscytotoxicity profiles comparable to cisplatin [Milovanovic M, DjekovicA, Volarevic V, Petrovic B, Arsenijevic N, Bugarcic Z D (2010) Ligandsubstitution reactions and cytotoxic properties of [Au(L)Cl₂]⁺ and[AuCl2(DMSO)₂]⁺ complexes (L=ethylenediamine and S-methyl-1-cysteine). JInorg Biochem 104:944-949—incorporated herein by reference in itsentirety]. In the present disclosure, a new series of gold(III)complexes 1-3 is developed by replacing two monodentate Cl⁻ ligands withbidentate 1,2-DACH (1,2-diaminocyclohexane) ligand and subjected to invitro cytotoxic evaluation against PC3 and SGC7901 cancer cell lines.

The in vitro cytotoxic effect of mixed ligand gold(III) diaminecomplexes against androgen-resistant prostate PC3 and human gastricSGC7901 cancer cells were studied using MTT assay. The in vitrocytotoxic activity depends on the exposure time and the concentration ofcomplexes. For that reason, different concentrations and a 3-dayexposure protocol were used in the present disclosure to determine theIC₅₀ values for all three complexes. The in vitro cytotoxicity in termsof IC₅₀ values of cisplatin for PC3 and SGC7901 cells was included for acomparison. The IC₅₀ data for the Au(III) complexes 1, 2 and 3 showed invitro cytotoxicity in a wide range of 1.1-8.9 IM for PC3 cells, as givenin Table 10. It can be seen from the IC₅₀ data for PC3 cancer cells thatcomplex 1 showed ca. 100% and 50% better potency than complexes 2 and 3,respectively. It can be concluded that complexes 1 is relatively moreeffective cytotoxic agent than complexes 2 and 3). For PC3 cancer cells,the order of in vitro cytotoxicity in terms of IC₅₀ values is cisplatin(1.1 μM)>complex 1 (4.8 μM)>complex 3 (6.1 μM)>complex 2 (8.9 μM), as itis known that the lower the IC₅₀ value is, the higher the in vitrocytotoxicity.

All three complexes showed slightly lower potency vis-a-vis cisplatin.According to IC₅₀ data of monim-al-Mehboob et al., [Au(en)₂]Cl₃ withethylenediamine ligands is a more prospective anti-cancer agent againstprostate cancer PC3 cells. The dose dependent studies showed that[Au(en)₂]Cl₃ was found to execute a powerful and promising cytotoxiceffect on PC3 cells which is comparable to that of cisplatin[Monim-ul-Mehboob M, Altaf M, Fettouhi M, Isab A A, Wazeer M I M, ShaikhM N, Altuwaijri S (2013) Synthesis, spectroscopic characterization andanti-cancer properties of new gold(III)-alkanediamine complexes againstgastric, prostate and ovarian cancer cells; crystal structure of[Au₂(pn)₂(Cl)₂]Cl₂.H₂O. Polyhedron 61:225-234—incorporated herein byreference in its entirety]. For PC3 cells, [Au(en)₂]Cl₃ was recognizedas effective cytotoxic agent as cisplatin while [(en)AuCl₂]Cl showedalmost 7-9 fold lower cytotoxicity as compared to cisplatin [Isab A A,Shaikh M N, Monim-ul-Mehboob M, Al-Maythalony B A, Wazeer M I M,Altuwaijri S (2011) Synthesis, characterization and antiproliferativeeffect of [Au(en)₂]Cl₃ and [Au(N-propyl-en)₂]Cl₃ on human cancer celllines. Spectrochim Acta (A) 79:1196-1201; Monim-ul-Mehboob M, Altaf M,Fettouhi M, Isab A A, Wazeer M I M, Shaikh M N, Altuwaijri S (2013)Synthesis, spectroscopic characterization and anti-cancer properties ofnew gold(III)-alkanediamine complexes against gastric, prostate andovarian cancer cells; crystal structure of [Au₂(pn)₂(Cl)₂]Cl₂.H₂O.Polyhedron 61:225-234—each incorporated herein by reference in itsentirety].

The IC₅₀ data for the Au(III) complexes 1, 2 and 3 showed in vitrocytotoxicity in the range of 5.5-7.9 IM for SGC7901 cells, as given inTable 10. It can be seen from the from IC₅₀ data for SGC7901 cancercells that complex 1 showed comparable in vitro cytotoxicity to complex3. Both complexes 2 and 3 are reasonably better cytotoxic agent thancomplex 3 For SGC7901 cancer cells, the order of in vitro cytotoxicityin terms of IC₅₀ values is complex 1 (5.5 μM)>complex 3 (5.8μM)>cisplatin (7.3 μM)>complex 2 (7.9 μM). It is worth-mentioning thatthe in vitro cytotoxicity of both complexes 1 and 3 are fairly betterthan that of cisplatin.

For SGC7901 cells, [Au(en)₂]Cl₃ show slightly lower cytotoxicity withrespect to cisplatin whereas [(en)AuCl₂]Cl almost two fold morecytotoxic than cisplatin [Monim-ul-Mehboob M, Altaf M, Fettouhi M, IsabA A, Wazeer M I M, Shaikh M N, Altuwaijri S (2013) Synthesis,spectroscopic characterization and anti-cancer properties of newgold(III)-alkanediamine complexes against gastric, prostate and ovariancancer cells; crystal structure of [Au₂(pn)₂(Cl)₂]Cl₂.H₂O. Polyhedron61:225-234—incorporated herein by reference in its entirety].[(en)AuCl₂]Cl may be potential anti-cancer agents for cisplatinresistant SCG7901 cells [Isab A A, Shaikh M N, Monim-ul-Mehboob M,Al-Maythalony B A, Wazeer M I M, Altuwaijri S (2011) Synthesis,characterization and antiproliferative effect of [Au(en)₂]Cl₃ and[Au(N-propyl-en)₂]Cl₃ on human cancer cell lines. Spectrochim Acta (A)79:1196-1201; Monim-ul-Mehboob M, Altaf M, Fettouhi M, Isab A A, WazeerM I M, Shaikh M N, Altuwaijri S (2013) Synthesis, spectroscopiccharacterization and anti-cancer properties of newgold(III)-alkanediamine complexes against gastric, prostate and ovariancancer cells; crystal structure of [Au₂(pn)₂(Cl)₂]Cl₂.H₂O. Polyhedron61:225-234—each incorporated herein by reference in its entirety]. Anindependent assessment of [Au(en)₂]Cl₃ and its derivatives reveals aninteresting feature that SGC7901 gastric cancer cells exhibit 7-8 foldintrinsic resistance relative to the PC3 cancer cells with respect tocisplatin. On the contrary, the [Au(en)2]Cl₃-type complexes may have thepotential to overcome mechanisms inducing resistance to cisplatin,particularly in the gastric cancer SGC7901 cells [Isab A A, Shaikh M N,Monim-ul-Mehboob M, Al-Maythalony B A, Wazeer M I M, Altuwaijri S (2011)Synthesis, characterization and antiproliferative effect of [Au(en)₂]Cl₃and [Au(N-propyl-en)₂]Cl₃ on human cancer cell lines. Spectrochim Acta(A) 79:1196-1201; Monim-ul-Mehboob M, Altaf M, Fettouhi M, Isab A A,Wazeer M I M, Shaikh M N, Altuwaijri S (2013) Synthesis, spectroscopiccharacterization and anti-cancer properties of newgold(III)-alkanediamine complexes against gastric, prostate and ovariancancer cells; crystal structure of [Au₂(pn)₂(Cl)₂]Cl₂.H₂O. Polyhedron61:225-234—each incorporated herein by reference in its entirety].Nevertheless, only two-fold or less resistance to the [Au(en)₂]Cl₃-typecomplexes was observed for PC3. This suggests that the intrinsic factorsregulating cellular sensitivity to cisplatin and [Au(en)₂]Cl₃ aredifferent for PC3 and SGC7901 cells. The factors affecting sensitivityof PC3 and SGC7901 towards cisplatin cells are analogous in the[Au(en)₂]Cl₃ type complexes [Isab A A, Shaikh M N, Monim-ul-Mehboob M,Al-Maythalony B A, Wazeer M I M, Altuwaijri S (2011) Synthesis,characterization and antiproliferative effect of [Au(en)₂]Cl₃ and[Au(N-propyl-en)₂]Cl₃ on human cancer cell lines. Spectrochim Acta (A)79:1196-1201; Monim-ul-Mehboob M, Altaf M, Fettouhi M, Isab A A, WazeerM I M, Shaikh M N, Altuwaijri S (2013) Synthesis, spectroscopiccharacterization and anti-cancer properties of newgold(III)-alkanediamine complexes against gastric, prostate and ovariancancer cells; crystal structure of [Au₂(pn)₂(CI)₂]Cl₂.H₂O. Polyhedron61:225-234—each incorporated herein by reference in its entirety]. Thesein vitro cytotoxicity results reveal that gold(III) complexes containingethylenediamine and 1,2-diaminocyclohexane ligands are better anticanceragents than [Au(1,2-DACH)Cl2]Cl, [Au(1,2-DACH)₂]Cl₃; and [Au(en)₂]Cl₃and its derivative complexes against gastric SCG7901 cancer cell line[Isab A A, Shaikh M N, Monim-ul-Mehboob M, Al-Maythalony B A, Wazeer M IM, Altuwaijri S (2011) Synthesis, characterization and antiproliferativeeffect of [Au(en)₂]Cl₃ and [Au(N-propyl-en)₂]Cl₃ on human cancer celllines. Spectrochim Acta (A) 79:1196-1201; Monim-ul-Mehboob M, Altaf M,Fettouhi M, Isab A A, Wazeer M I M, Shaikh M N, Altuwaijri S (2013)Synthesis, spectroscopic characterization and anti-cancer properties ofnew gold(III)-alkanediamine complexes against gastric, prostate andovarian cancer cells; crystal structure of [Au₂(pn)₂(Cl)₂]C₂.H₂O.Polyhedron 61:225-234—each incorporated herein by reference in itsentirety]; Al-Maythalony B A, Wazeer M I M, Isab A A (2009) Synthesisand characterization of gold(III) complexes with alkyldiamine ligands.Inorg Chim Acta 362:3109-3113; Al-Jaroudi S S, Fettouhi M, Wazeer M I M,Isab A A, Altuwaijri S (2013) Synthesis, characterization andcytotoxicity of new gold(III) complexes with 1,2-diaminocyclohexane:influence of stereochemistry on antitumor activity. Polyhedron50:434-442; Al-Jaroudi S S, Monim-ul-Mehboob M, Altaf M, Fettouhi M,Wazeer M I M, Isab A A (2014) Synthesis, spectroscopic characterization,X-ray structure and electrochemistry of new bis(1,2-diaminocyclohexane)gold(III) chloride compounds and their anticancer activities against PC3and SGC7901 cancer cell lines. New J Chem 38:3199-3211)—eachincorporated herein by reference in its entirety]. According to IC₅₀data presented herein, gold(III) complexes 1 and 3 were more effectivethan [Au(1,2-DACH)Cl₂]Cl against prostate PC3 cancer cells [Al-Jaroudi SS, Fettouhi M, Wazeer M I M, Isab A A, Altuwaijri S (2013) Synthesis,characterization and cytotoxicity of new gold(III) complexes with1,2-diaminocyclohexane: influence of stereochemistry on antitumoractivity. Polyhedron 50:434-442—incorporated herein by reference in itsentirety].

Thus, the foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. As will be understood by thoseskilled in the art, the present invention may be embodied in otherspecific forms without departing from the spirit or essentialcharacteristics thereof. Accordingly, the disclosure of the presentinvention is intended to be illustrative, but not limiting of the scopeof the invention, as well as other claims. The disclosure, including anyreadily discernible variants of the teachings herein, defines, in part,the scope of the foregoing claim terminology such that no inventivesubject matter is dedicated to the public.

1. (canceled) 2: The method of claim 9 wherein the compound of formula(A) is a gold(III) complex wherein: R₁-R₈ are each independentlyselected from the group consisting of a hydrogen atom, a substituted orunsubstituted methyl group, a substituted or unsubstituted ethyl group,a substituted or unsubstituted propyl group, a substituted orunsubstituted isopropyl group, a substituted or unsubstituted n-butylgroup, a substituted or unsubstituted isobutyl group, a substituted orunsubstituted sec-butyl group, a substituted or unsubstituted tert-butylgroup, a substituted or unsubstituted n-pentyl group, a substituted orunsubstituted neopentyl group, a substituted or unsubstituted sec-pentylgroup, a substituted or unsubstituted tert-pentyl group, a substitutedor unsubstituted n-hexane group, a substituted or unsubstitutedisohexane group, and a substituted or unsubstituted neohexane group; andR₉-R₂₀ are each independently a hydrogen atom, a halogen atom, aN-monosubstituted amino group, a N,N-disubstituted amino group, asubstituted or unsubstituted methyl group, a substituted orunsubstituted ethyl group, a substituted or unsubstituted propyl group,a substituted or unsubstituted isopropyl group, a substituted orunsubstituted n-butyl group, a substituted or unsubstituted isobutylgroup, a substituted or unsubstituted sec-butyl group, a substituted orunsubstituted tert-butyl group, a substituted or unsubstituted n-pentylgroup, a substituted or unsubstituted neopentyl group, a substituted orunsubstituted sec-pentyl group, a substituted or unsubstitutedtert-pentyl group, a substituted or unsubstituted n-hexane group, asubstituted or unsubstituted isohexane group, and a substituted orunsubstituted neohexane group. 3: The method of claim 9 wherein thecompound of formula (A) is a gold(III) complex having a formula selectedfrom the group consisting of Formula 1a, Formula 1b, Formula 2a andFormula 2b:

4: The method of claim 9 wherein the compound of formula (A) is agold(III) complex that comprises one or more pharmaceutically acceptableanions selected from the group consisting of fluoride, chloride,bromide, iodide, nitrate, sulfate, phosphate, amide, methanesulfonate,ethanesulfonate, p-toluenesulfonate, salicylate, malate, maleate,succinate, tartarate, citrate, acetate, perchlorate,trifluoromethanesulfonate, acetylacetonate, hexafluorophosphate, andhexafluoroacetylacetonate. 5-7. (canceled) 8: The method of claim 9,wherein the composition is formulated for one or more modes ofadministration selected from the group consisting of oraladministration, systemic administration, parenteral administration,inhalation spray, infusion, rectal administration, topicaladministration, intravesical administration, intradermal administration,transdermal administration, subcutaneous administration, intramuscularadministration, intralesional administration, intracranialadministration, intrapulmonal administration, intracardialadministration, intrasternal administration and sublingualadministration. 9: A method for treating prostate cancer and/orgastrointestinal cancer in a human in need thereof, comprising:administering a therapeutically effective amount of a compositioncomprising a compound of formula (A) and one or more pharmaceuticallyacceptable carriers,

or a pharmaceutically acceptable salt, ester, solvate or prodrugthereof; wherein the compound of formula (A) has a cis- ortrans-configuration; 1-6 each represents a carbon atom; R₁-R₈ are eachindependently selected from the group consisting of a hydrogen atom; alinear or branched, substituted or unsubstituted C₁-C₈ alkyl group; anda substituted or unsubstituted C₆-C₈ aryl group; and R9-R20 are eachindependently selected from the group consisting of a hydrogen atom, ahalogen atom, a hydroxyl group, a N-monosubstituted amino group, aN,N-disubstituted amino group, a substituted or unsubstituted C1-C8alkyl group, a substituted or unsubstituted C1-C8 alkoxy group asubstituted or unsubstituted C3-C8 cycloalkyl group, and a substitutedor unsubstituted C6-C8 aryl group; to the human suffering from theprostate cancer and/or gastrointestinal cancer mammalian subject. 10.(canceled) 11: The method of claim 9, wherein the therapeuticallyeffective amount comprises 5-50 μM of the gold(III) complex or apharmaceutically acceptable salt, ester, solvate or prodrug thereof.12-16. (canceled)